Selected reaction monitoring assays

ABSTRACT

Provided herein are methods for developing selected reaction monitoring mass spectrometry (LC-SRM-MS) assays.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/725,098 filed Dec. 21, 2012, which in turn claims priority andbenefit of U.S. Provisional Application No. 61/578,718 filed Dec. 21,2011, and U.S. Provisional Application No. 61/614,818 filed Mar. 23,2012, the contents of each of which are incorporated herein by referencein their entireties.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “IDIA-004_C01US_ST25.txt”, which wascreated on Jun. 11, 2015 and is 395,000 bytes in size, are herebyincorporated by reference in their entireties.

BACKGROUND

Liquid Chromatography Selected Reaction Monitoring Mass Spectrometry(LC-SRM-MS) has emerged as an alternative technology to immunoassays forquantification of target proteins in biological samples. LC-SRM-MSmethods are highly desirable because LC-SRM-MS methods provide bothabsolute structural specificity for the target protein and relative orabsolute measurement of the target protein concentration when suitableinternal standards are utilized. In contrast to immunoassays, LC-SRM-MSdoes not involve the manufacturing of biologics. LC-SRM-MS proteinassays can be rapidly and inexpensively developed in contrast to thedevelopment of immunoassays. LC-SRM-MS are highly multiplexed, withsimultaneous assays for hundreds of proteins performed in a singlesample analysis. Using LC-SRM-MS in contrast to other proteomictechnologies allows for complex assays for the identification diagnosticproteins in complex diseases such as cancer, autoimmune, and metabolicdisease. In particular, the development of a highly multiplexedLC-SRM-MS assay that reproducibly identifies a specific set of proteinsrelevant to a clinical disease presents diagnostic advantages andefficiencies. To date, proteomic techniques have not enabled suchinventions to exist where hundreds of proteins can be accuratelyquantified within a single sample. The present invention providesaccurate measurement of hundreds of lung cancer associated proteinswithin a single sample using multiplexed techniques.

SUMMARY OF THE INVENTION

The present invention comprises a LC-SRM-MS assay for the measurementproteins in a single sample and in a single LC-SRM-MS assay. The assaywas optimized for protein quantification and minimal interference amongproteins in the assay. This LC-SRM-MS assay is novel because measurementof a large number of proteins in a single sample specifically associatedwith lung cancer has not been accomplished. Simultaneous measurement ofsuch a large number of proteins without interference among the proteinsrequires specific techniques to distinguish among the proteins. Thecurrent invention provides clinical utility as this assay was used fordevelopment of lung cancer diagnostic tests for the early detection oflung cancer, managing disease treatment, as well as testing for diseaserecurrence.

The object of the present invention is to provide improved methods forthe use of LC-SRM-MS in the development of assays. Accordingly, providedherein is a method for developing peptides and transitions for aplurality of at least 200 proteins for a single sample selected reactionmonitoring mass spectrometry (LC-SRM-MS) assay, including the steps ofproviding a set of 200 or more proteins; generating transitions for eachprotein; assessing LC-SRM-MS data by Mascot score; performing collisionenergy optimization on the transitions; selecting peptides withtransitions showing the greatest peak areas of their transitions;selecting a set of transitions for each peptide, wherein the transitionsfor each peptide have one of the four most intense b or y transitionions; the transitions for each peptide have m/z values of at least 30m/z above or below those of the precursor ion; the transitions for eachpeptide do not interfere with transitions from other peptides; and thetransitions represent transitions due to breakage of peptide bond atdifferent sites of the protein.

In one embodiment of the method, each selected peptide in the set ofpeptides has a monoisotopic mass of 700-5000 Da; and does not contain acysteine or a methionine; or may contain cysteine or methionine. Inanother embodiment, the transitions for each peptide have one of thefour most intense b or y transition ions; have m/z values of at least 30m/z above or below those of a precursor ion; do not interfere withtransitions from other peptides; and represent transitions due tobreakage of peptide bond at different sites of the protein.

In another embodiment of the method, the peptides do not include anypeptide that is bounded by KK, KR, RK or RR (either upstream ordownstream) in the corresponding protein sequence. Specifically, theamino acid is charged at pH 7.0. These amino acids include arginine andlysine. In another embodiment, each peptide of said set of peptides isunique to the corresponding protein. In yet another embodiment, thepeptides do not include peptides which were observed inpost-translational modified forms. In still another embodiment, each setof peptides is prioritized according to one or more of the followingordered set of criteria: unique peptides first, then non-unique;peptides with no observed post-translational modifications first, thenthose observed with post-translational modifications; peptides withinthe mass range 800-3500 Da first, then those outside of 800-3500 Da; andsorted by decreasing number of variant residues. In certain embodiments,The peptides are unique in that they only appear once among the peptidesrun in a single assay.

In one embodiment, each set of peptides is prioritized according to allof the ordered set of criteria. In another embodiment, each prioritizedset of peptides contains 1-5 peptides.

In certain embodiments of the preceding methods, the two best peptidesper protein and the two best transitions per peptide are selected basedon experimental data resulting from LC-SRM-MS analysis of one or more ofthe following experimental samples: a biological disease sample, abiological control sample, and a mixture of synthetic peptides ofinterest. In a particular embodiment, the biological disease andbiological control samples are processed using an immunodepletion methodprior to LC-SRM-MS analysis. In another embodiment, the experimentalsamples contain internal standard peptides. In yet another embodiment,the LC-SRM-MS analysis method specifies a maximum of 7000 transitions,including transitions of the internal standard peptides and transitions.In other embodiments the method specifies a maximum of between1000-7000, 2000-6000, 3000-5000 and about 3500 transitions.

In one embodiment of the method, the top two transitions per peptide areselected according to one or more of the following criteria thetransitions exhibit the largest peak areas measured in either of the twobiological experimental samples; the transitions are not interfered withby other ions; the transitions do not exhibit an elution profile thatvisually differs from those of other transitions of the same peptide; orthe transitions are not beyond the detection limit of both of the twobiological experimental samples.

In another embodiment of the method, the top two peptides per proteinare selected according to one or more of the following criteria: one ormore peptides exhibit two transitions and represent the largest combinedpeak areas of the two transitions; or one or more peptides exhibit onetransition and represent the largest combined peak areas of the twotransitions.

In another aspect, provided herein is an assay developed according tothe foregoing method, and embodiments thereof.

In yet another aspect provided herein is the use of an assay developedaccording to the foregoing method, and embodiments thereof, to detect aplurality of at least 200 proteins in a single biological sample.

In another aspect, provided herein is an assay developed according tothe foregoing method, and embodiments thereof.

The disclosure provides a composition comprising at least fivetransition ions selected from the listing of transition ions in Table 2.In one embodiment of the assay each transition ion independentlycorresponds to a unique protein. The five transition ions correspondedto proteins selected from the group consisting of LRP1, BGH3, COIA1,TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, and GSLG1.(see: U.S. application Ser. No. 13/306,823 PCT/US11/62461). Thecomposition can further include an additional five transition ionsselected from the listing of transition ions in Table 2. The additionalfive transition ions can corresponded to the proteins APOE, BASP1, CD14,FOXA2 and HSPB1.

The disclosure provides a composition comprising at least five syntheticpeptides selected from the listing of peptides and proteins in Table 2.In one embodiment, each peptide can independently correspond to a uniqueprotein. At least one of the peptides was isotopically labeled. Theamount of each of the at least five synthetic peptides is known. Inanother embodiment, the composition included one or more polar solvents.The five synthetic peptides can correspond to the proteins LRP1, BGH3,COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, andGSLG1. The composition can also include an additional five syntheticpeptides selected from the listing of peptides and proteins in Table 2.The additional five synthetic peptides can correspond to the proteinsAPOE, BASP1, CD14, FOXA2 and HSPB1.

The disclosure provides a use of a composition, as described above, forthe development of an assay to detect a disease, disorder or conditionin a mammal.

The disclosure provides a method comprising analyzing a composition, asdescribed above, using mass spectrometry. The method can use selectedreaction monitoring mass spectrometry.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts candidate protein cohort by source. 217 tissue proteinswere identified using proteomics analysis. 319 proteins were identifiedby review of the literature. Between the two sources, there was anoverlap of 148 proteins.

FIG. 2 is a bar diagram showing Pearson correlations for peptides fromthe same peptide, from the same protein and from different proteins.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure relates to methods for developing peptides andtransitions for a single sample selected reaction monitoring massspectrometry (LC-SRM-MS) assay, generally comprising the steps ofproviding a set of proteins; identifying representative proteolyticpeptides for each protein according to a set of criteria; identifyingrepresentative transitions for each peptide according to another set ofcriteria; and selecting the optimum peptides per protein and the optimumtransitions per peptide.

Selected reaction monitoring mass spectrometry is capable of highlysensitive and accurate protein quantification based on thequantification of proteolytic peptides. In terms of clinical utility,mass spectrometry-based assays are often compared to immunoassays (e.g.,Enzyme-Linked Immunosorbent Assay, or ELISA), which have the ability toquantify specific analytes in large sample sets (e.g., 96 or 384 samplesin parallel microtitre plate-based format). Until recently, massspectrometry-based protein assays were not able to match these samplesizes or quantitative accuracy. Considerable time and expense isrequired to generate and characterize antibodies required forimmunoassays. Increasingly efficient LC-SRM-MS assays, therefore, maysurpass immunoassays such as ELISA in the rapid development ofclinically useful, multiplexed protein assays.

LC-SRM-MS is a highly selective method of tandem mass spectrometry whichhas the potential to effectively filter out all molecules andcontaminants except the desired analyte(s). This is particularlybeneficial if the analysis sample is a complex mixture which maycomprise several isobaric species within a defined analytical window.LC-SRM-MS methods may utilize a triple quadrupole mass spectrometerwhich, as is known in the art, includes three quadrupole rod sets. Afirst stage of mass selection is performed in the first quadrupole rodset, and the selectively transmitted ions are fragmented in the secondquadrupole rod set. The resultant transition (product) ions are conveyedto the third quadrupole rod set, which performs a second stage of massselection. The product ions transmitted through the third quadrupole rodset are measured by a detector, which generates a signal representativeof the numbers of selectively transmitted product ions. The RF and DCpotentials applied to the first and third quadrupoles are tuned toselect (respectively) precursor and product ions that have m/z valueslying within narrow specified ranges. By specifying the appropriatetransitions (m/z values of precursor and product ions), a peptidecorresponding to a targeted protein may be measured with high degrees ofsensitivity and selectivity. Signal-to-noise ratio in LC-SRM-MS is oftensuperior to conventional tandem mass spectrometry (MS/MS) experimentsthat do not selectively target (filter) particular analytes but ratheraim to survey all analytes in the sample.

Accordingly, provided herein is a method for developing peptides andtransitions for a plurality of proteins for use in selected reactionmonitoring mass spectrometry (LC-SRM-MS) assay. In a preferredembodiment, the assay involves the analysis of a single samplecontaining all analytes of interest (e.g., a proteolytic digest ofplasma proteins). As to the selection of the protease(s) used, trypsin,which cleaves exclusively C-terminal to arginine and lysine residues, isa preferred choice to generate peptides because the masses of generatedpeptides are compatible with the detection ability of most massspectrometers (up to 2000 m/z), the number and average length ofgenerated peptides, and also the availability of efficient algorithmsfor the generation of databases of theoretical trypsin-generatedpeptides. High cleavage specificity, availability, and cost are otheradvantages of trypsin. Other suitable proteases will be known to thoseof skill in the art. Miscleavage is a factor for failure or ambiguousprotein identification. A miscleavage can be defined as partialenzymatic protein cleavages generating peptides with internal missedcleavage sites reflecting the allowed number of sites (targeted aminoacids) per peptide that were not cut. The presence of post-translationalmodifications (PTMs) is also a potential contributor to the problem ofmiscleavages.

LC-SRM-MS mass spectrometry involves the fragmentation of gas phase ionsand occurs between the different stages of mass analysis. There are manymethods used to fragment the ions and these can result in differenttypes of fragmentation and thus different information about thestructure and composition of the molecule. The transition ions observedin an LC-SRM-MS spectrum result from several different factors, whichinclude, but are not limited to, the primary sequence, the amount ofinternal energy, the means of introducing the energy, and charge state.Transitions must carry at least one charge to be detected. An ion iscategorized as either a, b or c if the charge is on a transitioncomprising the original N terminus of the peptide, whereas the ion iscategorized as either x, y or z if the charge is on a transitioncomprising the original C terminus of the peptide. A subscript indicatesthe number of residues in the transition (e.g., one peptide residue inx₁, two peptide residues in y₂, and three peptide residues in z₃, etc.).

In a generic peptide repeat unit represented —N—C(O)—C—, an x ion and ana ion resulting from cleavage of the carbonyl-carbon bond (i.e.,C(O)—C). The x ion is an acylium ion, and the a ion is an iminium ion. Ay ion and a b ion result from cleavage of the carbonyl-nitrogen bond(i.e., C(O)—N, also known as the amide bond). In this case, the y ion isan ammonium ion and the b ion is an acylium ion. Finally, a z ion and ac ion result from cleavage of the nitrogen-carbon (i.e., C—N) bond. Thez ion is a carbocation and the c ion is an ammonium ion.

Superscripts are sometimes used to indicate neutral losses in additionto the backbone fragmentation, for example, * for loss of ammonia and^(∘) for loss of water. In addition to protons, c ions and y ions mayabstract an additional proton from the precursor peptide. Inelectrospray ionization, tryptic peptides may carry more than onecharge.

Internal transitions arise from double backbone cleavage. These may beformed by a combination of b-type and y-type cleavage (i.e., cleavageproducing b and y ions). Internal cleavage ions may also be formed by acombination of a-type and y-type cleavage. An internal transition with asingle side chain formed by a combination of a-type and y-type cleavageis called an iminium ion (sometimes also referred to as an imonium orimmonium ion). These ions are labeled with the one letter code for thecorresponding amino acid.

Low energy CID (i.e., collision induced dissociation in a triplequadrupole or an ion trap) involves the fragmentation of a peptidecarrying a positive charge, primarily along its backbone, to generateprimarily a, b and y ions.

In one aspect, provided herein is a method for developing peptides andtransitions for a plurality of proteins for a single sample selectedreaction monitoring mass spectrometry (LC-SRM-MS) assay, by: (a)providing a panel of a plurality of proteins; (b) identifying a set ofpeptides for each protein, wherein (i) each peptide in the set ofpeptides corresponds to a transition of said protein; (ii) the peptideshave a monoisotopic mass of 700-5000 Da; and (iii) the peptides do notcontain a cysteine or a methionine; or may contain cysteine ormethionine; (c) identifying a set of transitions for each peptide,wherein (i) the transitions for each peptide have one of the four mostintense b or y transition ions; (ii) the transitions for each peptidehave m/z values of at least 30 m/z above or below those of the precursorion; (iii) the transitions for each peptide do not interfere withtransitions from other peptides; and (iv) the transitions representtransitions due to breakage of peptide bond at different sites of theprotein; and (d) selecting the peptides for each protein that best fitthe criteria of step (b) and the transitions per peptide that best fitthe criteria of step (c); thereby developing peptides and transitionsfor a LC-SRM-MS assay.

By plurality of proteins it is meant at least 2, 3, 4, 5, 6, 7, 8, 9,10, 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500 or moreIn certain embodiments, the plurality of proteins can encompass between2 and 10. 10 and 20, 20 and 50, 50 and 100, 100 and 200, or 200 and 500proteins. In other embodiments, the plurality of proteins can encompassbetween 250 and 450; or 300 and 400 proteins.

Trypsin-like proteases cleave peptide bonds following a positivelycharged amino acid (e.g., lysine (K) or arginine (R)). This specificityis driven by the residue which lies at the base of the enzyme's S1pocket (generally a negatively charged aspartic acid or glutamic acid).Accordingly, in one embodiment of the method, the peptides do notinclude any peptide that is bounded by KK, KR, RK or RR, either upstreamof downstream in the corresponding protein sequence. In anotherembodiment, each peptide of said set of peptides is unique to thecorresponding protein.

Post-translational modification (PTM) is the chemical modification of aprotein after its translation. It can include any modification followingtranslation, including cleavage. It is one of the later steps in proteinbiosynthesis, and thus gene expression, for many proteins. It isdesirable to avoid such peptides for the purpose of proteinidentification. Thus, in another embodiment, the peptides do not includepeptides which were observed in post-translational modified forms.

In still another embodiment, each set of peptides is prioritizedaccording to one or more of the following ordered set of criteria: (a)unique peptides first, then non-unique; (b) peptides with no observedpost-translational modifications first, then those observed withpost-translational modifications; (c) peptides within the mass range800-3500 Da first, then those outside of 800-3500 Da; and (d) sorted bydecreasing number of variant residues. In one embodiment, each set ofpeptides is prioritized according to all of the ordered set of criteria.In another embodiment, each prioritized set of peptides contains 1-5peptides.

In certain embodiments, one or more liquid chromatography (LC)purification steps are performed prior to a subsequent LC-SRM-MSanalysis step. Traditional LC analysis relies on the chemicalinteractions between sample components and column packing materials,where laminar flow of the sample through the column is the basis forseparation of the analyte of interest from the test sample. The skilledartisan will understand that separation in such columns is a diffusionalprocess. A variety of column packing materials are available forchromatographic separation of samples, and selection of an appropriateseparation protocol is an empirical process that depends on the samplecharacteristics, the analyte of interest, the interfering substancespresent and their characteristics, etc. Various packing chemistries canbe used depending on the needs (e.g., structure, polarity, andsolubility of compounds being purified). In various embodiments thecolumns are polar, ion exchange (both cation and anion), hydrophobicinteraction, phenyl, C-2, C-8, C-18 columns, polar coating on porouspolymer, or others that are commercially available. Duringchromatography, the separation of materials is effected by variablessuch as choice of eluant (also known as a “mobile phase”), choice ofgradient elution and the gradient conditions, temperature, etc. Incertain embodiments, an analyte may be purified by applying a sample toa column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. As discussed above, suchprocedures may enrich the amount of one or more analytes of interestrelative to one or more other components of the sample.

The following parameters are used to specify an LC-SRM-MS assay of aprotein under a particular LC-SRM-MS system: (1) a tryptic peptide ofthe protein; (2) the retention time (RT) of the peptide; (3) the m/zvalue of the peptide precursor ion; (4) the declustering potential usedto ionize the precursor ion; (5) the m/z value of a fragment iongenerated from the peptide precursor ion; and (6) the collision energy(CE) used to fragment the peptide precursor ion that is optimized forthe particular peptide.

In certain embodiments of the preceding methods, the two best peptidesper protein and the two best transitions per peptide are selected basedon experimental data resulting from LC-SRM-MS analysis of one or more ofthe following experimental samples: a biological disease sample, abiological control sample, and a mixture of synthetic peptides ofinterest. Biological samples include body fluids, tissue samples andcell samples. Body fluid samples can include blood, serum, sputum,genital secretions, cerebrospinal fluid, sweat or excreta such as urine.Body tissue samples can include lung, skin, brain, spine, bone, muscle,epithelial, liver, kidney, pancreas, gastrointestinal tract,cardiovascular tissue, heart or nervous tissue. Biological diseasesamples can include cancer, benign tumors, infected tissue and tissuesubject to trauma. In a particular embodiment, the biological diseaseand biological control samples are processed using an immunodepletionmethod prior to LC-SRM-MS analysis. Immunodepletion involves removal ofone or more proteins through the use of antibodies. Numerousimmunodepletion techniques are known to those of skill in the art. Inanother embodiment, the biological disease and biological controlsamples are processed using an immunocapture method prior to LC-SRM-MSanalysis. Immunocapture involves selection of one or more proteinsthrough the use of antibodies. Numerous immunocapture techniques areknown to those of skill in the art.

To facilitate accurate quantification of the peptide transitions by themethods disclosed herein, a set of isotopically-labeled syntheticversions of the peptides of interest may be added in known amounts tothe sample for use as internal standards. Since the isotopically-labeledpeptides have physical and chemical properties identical to thecorresponding surrogate peptide, they co-elute from the chromatographiccolumn and are easily identifiable on the resultant mass spectrum. Theaddition of the labeled standards may occur before or after proteolyticdigestion. Methods of synthesizing isotopically-labeled peptides will beknown to those of skill in the art. Thus, in another embodiment, theexperimental samples contain internal standard peptides. Otherembodiments may utilize external standards or other expedients forpeptide quantification.

In yet another embodiment, the LC-SRM-MS analysis method specifies amaximum of 7000 transitions, including transitions of the internalstandard peptides and transitions. As used herein, the term “transition”refers to the specific pair of m/z (mass-to-charge) values associatedwith the precursor and transition ions corresponding to a specificpeptide and, therefore, to a specific protein.

In one embodiment of the method, the top two transitions per peptide areselected according to one or more of the following criteria (A): (1) thetransitions exhibit the largest peak areas measured in either of the twobiological experimental samples; (2) the transitions are not interferedwith by other ions; (3) the transitions do not exhibit an elutionprofile that visually differs from those of other transitions of thesame peptide; (4) the transitions are not beyond the detection limit ofboth of the two biological experimental samples; (5) the transitions donot exhibit interferences.

For the mass spectrometric analysis of a particular peptide, thequantities of the peptide transitions in the sample may be determined byintegration of the relevant mass spectral peak areas, as known in theprior art. When isotopically-labeled internal standards are used, asdescribed above, the quantities of the peptide transitions of interestare established via an empirically-derived or predicted relationshipbetween peptide transition quantity (which may be expressed asconcentration) and the area ratio of the peptide transition and internalstandard peaks at specified transitions.

In another embodiment of the method, the top two peptides per proteinare selected according to one or more of the following criteria (B): (1)one or more peptides exhibit two transitions according to criteria (A)and represent the largest combined peak areas of the two transitionsaccording to criteria (A); and (2) one or more peptides exhibit onetransition according to criteria (A) and represent the largest combinedpeak areas of the two transitions according to criteria (A).

Assays

The methods of the present disclosure allow the quantification of highabundance and low abundance plasma proteins that serve as detectablemarkers for various health states (including diseases and disorders),thus forming the basis for assays that can be used to determine thedifferences between normal levels of detectable markers and changes ofsuch detectable markers that are indicative of changes in health status.In one aspect of the invention, provided herein is an assay developedaccording to the foregoing method, and embodiments thereof. In anotheraspect, provided herein is the use of an assay developed according tothe foregoing method, and embodiments thereof, to detect a plurality ofat least 200, 300, or more proteins in a single sample. In a merelyillustration embodiment, 388 proteins in the following table 1 aredetected utilizing the method of present invention.

Of the 388 proteins, the 36 most cooperative proteins are listed inTable 2.

SRM assays for the 388 proteins were developed using standard syntheticpeptide techniques. Of the 388 candidates, SRM assays were successfullydeveloped for 371 candidates. The 371 SRM assays were applied to benignand lung cancer plasma samples to evaluate detection rate in blood. Thesummary of the SRM assay for these 371 proteins is listed in table 3(see also Example III).

DEFINITIONS

As used herein, “transition” refers to a pair of m/z values associatedwith a peptide. Normally, labeled synthetic peptides are used as qualitycontrols in SRM assays. However, for very large SRM assays such as the371 protein lung cancer assay, labeled peptides are not feasible.However, correlation techniques (Kearney, Butler et al. 2008) were usedto confirm the identity of protein transitions with high confidence. InFIG. 2 a histogram of the Pearson correlations between every pair oftransitions in the assay is presented. The correlation between a pair oftransitions is obtained from their expression profiles over all samples(143) in the training study detailed below. As expected, transitionsfrom the same peptide are highly correlated. Similarly, transitions fromdifferent peptide fragments of the same protein are also highlycorrelated. In contrast, transitions from different proteins are nothighly correlated. This methodology enables a statistical analysis ofthe quality of a protein's SRM assay. For example, if the correlation oftransitions from two peptides from the same protein is above 0.5 thenthere is less than a 5% probability that the assay is false.

As used herein, a “tryptic peptide” refers to the peptide that is formedby the treatment of a protein with trypsin.

As used herein, “RT” refers to “retention time”, the elapsed timebetween injection and elution of an analyte.

As used herein, “m/z” indicates the mass-to-charge ratio of an ion.

As used herein, “DP” refers to “declustering potential”, a voltagepotential to dissolvate and dissociate ion clusters. It is also known as“fragmentor voltage” or “ion transfer capillary offset voltage”depending on the manufacture.

As used herein, “CE” refers to “collision energy”, the amount of energyprecursor ions receive as they are accelerated into the collision cell.

As used herein, “LC-SRM-MS” is an acronym for “selected reactionmonitoring” and may be used interchangeably with “LC-MRM-MS”.

As used herein, “MS/MS” represents tandem mass spectrometry, which is atype of mass spectrometry involving multiple stages of mass analysiswith some form of fragmentation occurring in between the stages.

As used herein, “ISP” refers to “internal standard peptides”.

As used herein, “HGS” refers to “human gold standard”, which iscomprised of a pool of plasma from healthy individuals.

As used herein, “MGF” refers to “Mascot generic file”. Mascot is asearch engine that uses mass spectrometry data to identify proteins fromprimary sequence databases. A Mascot generic file is a plain text(ASCII) file containing peak list information and, optionally, searchparameters.

Mascot is a web-based tool for deriving protein sequences from massspectrometry data. This data can be acquired from any mass spectrometrytechnique including MALDI-TOF and MS/MS (including LC-SRM-MS) data.Mascot uses a ‘probability-based MOWSE’ algorithm to estimate thesignificance of a match (i.e., that the observed transitions correspondto a particular protein). The total score is the absolute probabilitythat the observed match is a random event. They are reported as −10×LOG10(P), where P is the absolute probability. Lower probabilities,therefore, are reported as higher scores. For example, if the absoluteprobability that an observed match is random is 1×10⁻¹², Mascot reportsit as 120.

The disclosure also provides compositions. These compositions caninclude any of the transition ions described in Table 2. Thesetransition ions exist while peptides derived from the proteins in Table2 are undergoing analysis with LC-SRM-MS. In one embodiment, thecomposition includes any of the transition ions described in Table 2. Inanother embodiment, the composition includes any two transition ionsdescribed in Table 2. In other embodiments, the composition includes,any 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,270, 280, 290, 300 or 331 transition ions described in Table 2.

In another embodiment, the transition ions correspond with humanproteins including LRP1, BGH3, COIA1, TETN, TSP1, ALDOA, GRP78, ISLR,FRIL, LG3BP, PRDX1, FIBA, and GSLG1. In another embodiment, thetransition ions are derived from human proteins including LRP1, BGH3,COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, andGSLG1. These proteins can further include transition ions correspondingwith and/or derived from any number of additional proteins from Table 2.Thus, the composition can include, any additional 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150,160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290,300 or 331 transition ions described in Table 2.

In another embodiment, each of the transition ions in the compositioncorresponds and/or is derived from a different protein. In anotherembodiment, 90% of the transition ions in the composition correspondwith and/or are derived from a protein that no other transition ion inthe composition corresponds. In other embodiments, 80, 70, 60, 50, 40,30, 20, 10 or 0% of the transition ions in the composition correspondand/or are derived from a protein that no other transition ion in thecomposition corresponds.

The compositions described herein included synthetic peptides. Syntheticpeptides can be used as controls for the abundance of proteins they arederived from and/or correspond. In certain embodiments, the abundance ofthe synthetic peptides is defined and the results are compared toLC-SRM-MS results from a peptide found in a sample to the LC-SRM-MSresults in the corresponding synthetic peptide. This allows for thecalculation of the abundance of the peptide in the sample. In certainembodiments, by knowing the abundance of a peptide in a sample, theabundance of the protein it corresponded to is determined.

Synthetic peptides can be generated using any method known in the art.These methods can include recombinant expression techniques such asexpression in bacteria or in vitro expression in eukaryotic cell lysate.These methods can also include solid phase synthesis.

In one embodiment, the composition includes synthetic peptides selectedfrom any of the peptides described in Table 2. In another embodiment,the composition included any two peptides described in Table 2. In otherembodiments, the composition included, any 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 or morepeptides described in Table 2.

In another embodiment, the peptides corresponded with human genesincluding LRP1, BGH3, COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL,LG3BP, PRDX1, FIBA, and GSLG1 as described in corresponding patentapplication. These genes can further include peptides corresponding withany number of additional genes from Table 2. Thus, the composition caninclude, any additional 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300 or more peptides describedin Table 2.

In another embodiment, each of the peptides in the composition eachcorresponds with a different protein. In another embodiment, 90% of thepeptides in the composition correspond with a protein that no otherpeptide in the composition corresponds with. In other embodiments, 80,70, 60, 50, 40, 30, 20, 10 or 0% of the peptides in the compositioncorrespond with from a protein that no other peptide in the compositioncorresponds with.

The peptides can be isotopically labeled. The isotopes with which theycan be labeled include ¹³C, ²H, ¹⁵N and ¹⁸O. The peptides can alsoinclude a polar solvent. Polar solvents can include water and mixturesof ethanol and water.

In certain embodiments, the samples described herein are taken frommammals. These mammals include rats, mice, rabbits, dogs, non-humanprimates and humans. Samples can be isolated from any tissue or organ orfrom any bodily fluid. Organs from which samples can be taken includeskin, heart, lung, brain, kidney, liver, pancreas, spleen, testes,ovaries, gall bladder, thymus, thyroid, eye, ear, nose, mouth, tongue,penis, vagina, bladder or larynx. Tissues include nervous tissue,vascular tissue, muscle, bone, gastrointestinal tract, epithelialtissue, fibroblastic tissue, mucous membranes, hair, skin, reproductivetissue and connective tissue. Body fluids and excretions include, blood,serum, saliva, urine, semen, vaginal secretions, excrement, bile, tears,lymph, ear wax, mucous, shed skin, finger nails, toe nails, skin oils,sweat and dandruff.

The relative abundance of one or more of the proteins represented by thetransition ions and synthetic peptides described above can be used todiagnose, determine likelihood of the presence of, develop prognoses forand/or stage various diseases and pathologies. Often the organ, tissueor bodily fluid or excretion from which the sample is taken is distinctfrom the organ, tissue or bodily fluid or excretion involved with thedisease or pathology. For example, the presence of lung cancer can bedetermined from a sample taken from blood. Any type of body fluid may beused in the assays.

Diseases and pathologies that status, diagnosis, presence or prognosiscan be found using the transition ions and/or synthetic peptidesdescribed herein include cancer, metabolic diseases, neurologicaldisorders, infectious diseases and cardiovascular disorders.

Examples Exemplary Standard Operating Procedure Protein Selection

Proteins known to be over-expressed on the cell surface of lung cancertumors were obtained (through literature searching, experimental data orproprietary databases). This was referred to as set ‘A’. Proteins knownto be over-secreted by lung cancer tumor cells were obtained (throughliterature searching, experimental data or proprietary databases). Thiswas referred to as set ‘B’. Proteins associated with lung cancer in theliterature were mined. This was referred to as set ‘C’. Proteins ofinterest (sets A, B and C are merged resulting in over 700 proteins)were assembled. The set of proteins was reduced to a set of 388 proteins(see Table 4) by prioritizing those proteins that have been previouslydetected my LC-MS/MS in blood (serum or plasma).

Selected proteins were then identified by their UniProt protein name andaccession, their Entrez gene symbol and gene name, the isoform accessionand their amino acid sequence. The canonical isoform in UniProt wasselected if a protein has more than one isoform.

Peptide Selection for Synthesis

The five best peptides per protein for LC-SRM-MS assay were selected foras follows. Fully tryptic peptides having a monoisotopic mass of800-3500 mass units, without miscleavages, not containing a cysteine (C)or a methionine (M), without having high miscleavage probability wereselected. Further, any peptide that was bounded by KK, KR, RK or RR(either upstream or downstream) in the corresponding protein sequencewas not selected.

Peptides were selected that were unique to the protein of interest.Peptides were only selected that match only one protein or proteinfamily including analogues of the one protein, when searched in proteindatabases. Further, peptides which were observed in post-translationalmodified forms were not selected. Databases were assessed that showedexpression of the proteins from which the peptides were isolated inhuman blood. Also databases of good quality MS peptides were searched.Peptides that appeared in human blood and were good quality MS peptideswere favored. If these methods did not result in a sufficient number ofpeptides, rules were relaxed in a step wise manner to allow a greaternumber of peptides until a sufficient number was reached. The purity ofthe synthesized peptides was >75% and the amount of material was ≧25 μg.Peptides did not need to be desalted.

The four best transitions per peptide are then selected and optimizedbased on experimental results from a mixture of synthetic peptides.LC-SRM-MS-triggered MS/MS spectra were acquired for each syntheticpeptide, using a QTRAP 5500 instrument. One spectrum for the doubly- andone for the triply-charged precursor ion was collected for each peptideFor the identified peptides (Mascot score≧15), retention time wasrecorded for the four most intense b or y transition ions. The selectedtransition ions possessed m/z values were at least 30 m/z above or belowthose of the precursor ions; they did not interfere with other syntheticpeptides; and they were transition ions due to breakage of peptide bondat different sites.

If an insufficient percentage of the synthetic peptides were acquired,the steps were repeated. In some cases, the second transition with firstwith theoretical y+ ions with m/z values at least 30 m/z above those ofthe doubly charged precursor ion was selected if an insufficientpercentage was acquired. Peptides that failed to trigger the acquisitionof MS/MS spectrum were discarded.

Collision energy (CE) for each selected transition (See Table 4) wasoptimized.

Exemplary Protein List

The abundance of the following proteins can be assessed substantiallysimultaneously using the MS-LC-SRM-MS system described herein.Transitions from these proteins can be used to diagnose diseasesincluding lung cancer when their abundance is measured in a biologicalspecimen from a subject to be diagnosed for lung cancer. In oneembodiment, the abundances of these proteins are measured in the bloodserum of the subject.

TABLE 1 Sources of Subcellular Evidence for Gene Tissue BiomarkersLocation Presence in UniProt Protein Protein Name Symbol Biomarkers inLiterature (UniProt) Blood 1433B_HUMAN 14-3-3 YWHAB Secreted,LungCancers Cytoplasm. Literature, protein EPI Melanosome. Detectionbeta/alpha Note = Identified by mass spectrometry in melanosomefractions from stage I to stage IV. 1433E_HUMAN 14-3-3 YWHAE ENDOLungCancers, Cytoplasm (By Literature, protein BenignNodulessimilarity). Detection epsilon Melanosome. Note = Identified by massspectrometry in melanosome fractions from stage I to stage IV.1433S_HUMAN 14-3-3 SFN Secreted, LungCancers Cytoplasm. UniProt, proteinEPI Nucleus (By Literature, sigma similarity). Detection Secreted. Note= May be secreted by a non-classical secretory pathway. 1433T_HUMAN14-3-3 YWHAQ EPI LungCancers, Cytoplasm. Detection protein BenignNodulesNote = In theta neurons, axonally transported to the nerve terminals.1433Z_HUMAN 14-3-3 YWHAZ EPI LungCancers, Cytoplasm. Detection proteinBenignNodules Melanosome. zeta/delta Note = Located to stage I to stageIV melanosomes. 6PGD_HUMAN 6-phosphogluconate PGD EPI, ENDO Cytoplasm(By Detection dehydrogenase, similarity). decarboxylating A1AG1_HUMANAlpha-1- ORM1 EPI Symptoms Secreted. UniProt, acid Literature,glycoprotein 1 Detection, Prediction ABCD1_HUMAN ATP- ABCD1 ENDOPeroxisome Detection, binding membrane; Prediction cassette Multi-passsub-family membrane D member 1 protein. ADA12_HUMAN Disintegrin ADAM12LungCancers, Isoform 1: Cell UniProt, and BenignNodules, membrane;Detection, metalloproteinase Symptoms Single-pass Prediction domain-type I containing membrane protein 12 protein.|Isoform 2:Secreted.|Isoform 3: Secreted (Potential).|Isoform 4: Secreted(Potential). ADML_HUMAN ADM ADM LungCancers, Secreted. UniProt,BenignNodules, Literature, Symptoms Detection, Prediction AGR2_HUMANAnterior AGR2 EPI LungCancers Secreted. UniProt, gradient EndoplasmicPrediction protein 2 reticulum (By homolog similarity). AIFM1_HUMANApoptosis- AIFM1 EPI, ENDO LungCancers Mitochondrion Detection, inducingintermembrane Prediction factor 1, space. Nucleus. mitochondrial Note =Translocated to the nucleus upon induction of apoptosis. ALDOA_HUMANFructose- ALDOA Secreted, LungCancers, Literature, bisphosphate EPISymptoms Detection aldolase A AMPN_HUMAN Aminopeptidase N ANPEP EPI,ENDO LungCancers, Cell membrane; UniProt, BenignNodules, Single-passDetection Symptoms type II membrane protein. Cytoplasm, cytosol(Potential). Note = A soluble form has also been detected. ANGP1_HUMANAngiopoietin-1 ANGPT1 LungCancers, Secreted. UniProt, BenignNodulesLiterature, Prediction ANGP2_HUMAN Angiopoietin-2 ANGPT2 LungCancers,Secreted. UniProt, BenignNodules Literature, Prediction APOA1_HUMANApolipoprotein A-I APOA1 LungCancers, Secreted. UniProt, BenignNodules,Literature, Symptoms Detection, Prediction APOE_HUMAN Apolipoprotein EAPOE EPI, ENDO LungCancers, Secreted. UniProt, BenignNodules,Literature, Symptoms Detection, Prediction ASM3B_HUMAN Acid SMPDL3B EPI,ENDO Secreted (By UniProt, sphingomyelinase-like similarity). Predictionphosphodiesterase 3b AT2A2_HUMAN Sarcoplasmic/ ATP2A2 EPI, ENDOLungCancers, Endoplasmic Detection endoplasmic BenignNodules reticulumreticulum membrane; calcium Multi-pass ATPase 2 membrane protein.Sarcoplasmic reticulum membrane; Multi-pass membrane protein. ATS1_HUMANA ADAMTS1 LungCancers, Secreted, UniProt, disintegrin BenignNodules,extracellular Literature, and Symptoms space, Predictionmetalloproteinase with extracellular thrombospondin matrix (By motifs 1similarity). ATS12_HUMAN A ADAMTS12 LungCancers Secreted, UniProt,disintegrin extracellular Detection, and space, Predictionmetalloproteinase with extracellular thrombospondin matrix (By motifs 12similarity). ATS19_HUMAN A ADAMTS19 LungCancers Secreted, UniProt,disintegrin extracellular Prediction and space, metalloproteinase withextracellular thrombospondin matrix (By motifs 19 similarity).BAGE1_HUMAN B BAGE LungCancers Secreted UniProt, melanoma (Potential).Prediction antigen 1 BAGE2_HUMAN B BAGE2 LungCancers Secreted UniProt,melanoma (Potential). Prediction antigen 2 BAGE3_HUMAN B BAGE3LungCancers Secreted UniProt, melanoma (Potential). Prediction antigen 3BAGE4_HUMAN B BAGE4 LungCancers Secreted UniProt, melanoma (Potential).Prediction antigen 4 BAGE5_HUMAN B BAGE5 LungCancers Secreted UniProt,melanoma (Potential). Prediction antigen 5 BASP1_HUMAN Brain acid BASP1Secreted, Cell membrane; Detection soluble EPI Lipid-anchor. protein 1Cell projection, growth cone. Note = Associated with the membranes ofgrowth cones that form the tips of elongating axons. BAX_HUMAN ApoptosisBAX EPI LungCancers, Isoform Alpha: UniProt, regulator BenignNodulesMitochondrion Literature, BAX membrane; Prediction Single-pass membraneprotein. Cytoplasm. Note = Colocalizes with 14- 3-3 proteins in thecytoplasm. Under stress conditions, redistributes to the mitochondrionmembrane through the release from JNK- phosphorylated 14-3-3proteins.|Isoform Beta: Cytoplasm.|Isoform Gamma: Cytoplasm.|IsoformDelta: Cytoplasm (Potential). BDNF_HUMAN Brain- BDNF BenignNodules,Secreted. UniProt, derived Symptoms Literature, neurotrophic Predictionfactor BGH3_HUMAN Transforming TGFBI LungCancers, Secreted, UniProt,growth BenignNodules extracellular Detection factor-beta- space, inducedextracellular protein ig- matrix. h3 Note = May be associated both withmicrofibrils and with the cell surface. BMP2_HUMAN Bone BMP2LungCancers, Secreted. UniProt, morphogenetic BenignNodules, Literatureprotein 2 Symptoms BST1_HUMAN ADP- BST1 EPI Symptoms Cell membrane;Detection, ribosyl Lipid-anchor, Prediction cyclase 2 GPI-anchor.C163A_HUMAN Scavenger CD163 EPI Symptoms Soluble CD163: UniProt,receptor Secreted.|Cell Detection cysteine- membrane; rich type 1Single-pass protein type I M130 membrane protein. Note = Isoform 1 andisoform 2 show a lower surface expression when expressed in cells.C4BPA_HUMAN C4b- C4BPA LungCancers, Secreted. UniProt, binding SymptomsDetection, protein Prediction alpha chain CAH9_HUMAN Carbonic CA9LungCancers, Nucleus. UniProt anhydrase BenignNodules, Nucleus, 9Symptoms nucleolus. Cell membrane; Single-pass type I membrane protein.Cell projection, microvillus membrane; Single-pass type I membraneprotein. Note = Found on the surface microvilli and in the nucleus,particularly in nucleolus. CALR_HUMAN Calreticulin CALR EPI SymptomsEndoplasmic UniProt, reticulum Literature, lumen. Detection, Cytoplasm,Prediction cytosol. Secreted, extracellular space, extracellular matrix.Cell surface. Note = Also found in cell surface (T cells), cytosol andextracellular matrix. Associated with the lytic granules in thecytolytic T- lymphocytes. CALU_HUMAN Calumenin CALU EPI SymptomsEndoplasmic UniProt, reticulum Detection, lumen. Prediction Secreted.Melanosome. Sarcoplasmic reticulum lumen (By similarity). Note =Identified by mass spectrometry in melanosome fractions from stage I tostage IV. CALX_HUMAN Calnexin CANX Secreted, BenignNodules EndoplasmicUniProt, EPI, ENDO reticulum Literature, membrane; Detection Single-passtype I membrane protein. Melanosome. Note = Identified by massspectrometry in melanosome fractions from stage I to stage IV.CAP7_HUMAN Azurocidin AZU1 EPI Symptoms Cytoplasmic Prediction granule.Note = Cytoplasmic granules of neutrophils. CATB_HUMAN Cathepsin CTSBSecreted LungCancers Lysosome. Literature, B Melanosome. Detection, Note= Identified Prediction by mass spectrometry in melanosome fractionsfrom stage I to stage IV. CATG_HUMAN Cathepsin CTSG Secreted,BenignNodules Cell surface. Detection, G ENDO Prediction CBPB2_HUMANCarboxypeptidase CPB2 LungCancers, Secreted. UniProt, B2 BenignNodules,Detection, Symptoms Prediction CCL22_HUMAN C-C motif CCL22 LungCancers,Secreted. UniProt, chemokine 22 BenignNodules Prediction CD14_HUMANMonocyte CD14 EPI LungCancers, Cell membrane; Literature,differentiation BenignNodules, Lipid-anchor, Detection, antigen SymptomsGPI-anchor. Prediction CD14 CD24_HUMAN Signal CD24 LungCancers, Cellmembrane; Literature transducer BenignNodules Lipid-anchor, CD24GPI-anchor. CD2A2_HUMAN Cyclin- CDKN2A LungCancers, Cytoplasm.Literature, dependent BenignNodules Nucleus.|Nucleus, Prediction kinasenucleolus inhibitor (By similarity). 2A, isoform 4 CD38_HUMAN ADP- CD38EPI, ENDO Symptoms Membrane; UniProt, ribosyl Single-pass Literaturecyclase 1 type II membrane protein. CD40L_HUMAN CD40 CD40LG LungCancers,Cell membrane; UniProt, ligand BenignNodules, Single-pass LiteratureSymptoms type II membrane protein.|CD40 ligand, soluble form: Secreted.CD44_HUMAN CD44 CD44 EPI LungCancers, Membrane; UniProt, antigenBenignNodules, Single-pass Literature, Symptoms type I Detection,membrane Prediction protein. CD59_HUMAN CD59 CD59 LungCancers, Cellmembrane; UniProt, glycoprotein BenignNodules, Lipid-anchor, Literature,Symptoms GPI-anchor. Detection, Secreted. Prediction Note = Soluble formfound in a number of tissues. CD97_HUMAN CD97 CD97 EPI, ENDO SymptomsCell membrane; UniProt antigen Multi-pass membrane protein.|CD97 antigensubunit alpha: Secreted, extracellular space. CDCP1_HUMAN CUB CDCP1LungCancers Isoform 1: Cell UniProt, domain- membrane; Predictioncontaining Single-pass protein 1 membrane protein (Potential). Note =Shedding may also lead to a soluble peptide.|Isoform 3: Secreted.CDK4_HUMAN Cell CDK4 LungCancers, Literature division Symptoms proteinkinase 4 CEAM5_HUMAN Carcinoembryonic CEACAM5 EPI LungCancers, Cellmembrane; Literature, antigen- BenignNodules, Lipid-anchor, Predictionrelated cell Symptoms GPI-anchor. adhesion molecule 5 CEAM8_HUMANCarcinoembryonic CEACAM8 EPI LungCancers Cell membrane; Detection,antigen- Lipid-anchor, Prediction related cell GPI-anchor. adhesionmolecule 8 CERU_HUMAN Ceruloplasmin CP EPI LungCancers, Secreted.UniProt, Symptoms Literature, Detection, Prediction CH10_HUMAN 10 kDaHSPE1 ENDO LungCancers Mitochondrion Literature, heat shock matrix.Detection, protein, Prediction mitochondrial CH60_HUMAN 60 kDa HSPD1Secreted, LungCancers, Mitochondrion Literature, heat shock EPI, ENDOSymptoms matrix. Detection protein, mitochondrial CKAP4_HUMANCytoskeleton- CKAP4 EPI, ENDO LungCancers Endoplasmic UniProt associatedreticulum-Golgi protein 4 intermediate compartment membrane; Single-passmembrane protein (Potential). CL041_HUMAN Uncharacterized C12orf41 ENDOPrediction protein C12orf41 CLCA1_HUMAN Calcium- CLCA1 LungCancers,Secreted, UniProt, activated BenignNodules extracellular Predictionchloride space. Cell channel membrane; regulator 1 Peripheral membraneprotein; Extracellular side. Note = Protein that remains attached to theplasma membrane appeared to be predominantly localized to microvilli.CLIC1_HUMAN Chloride CLIC1 EPI Nucleus. UniProt, intracellular NucleusLiterature, channel membrane; Detection protein 1 Single-pass membraneprotein (Probable). Cytoplasm. Cell membrane; Single-pass membraneprotein (Probable). Note = Mostly in the nucleus including in thenuclear membrane. Small amount in the cytoplasm and the plasma membrane.Exists both as soluble cytoplasmic protein and as membrane protein withprobably a single transmembrane domain. CLUS_HUMAN Clusterin CLU EPI,ENDO LungCancers, Secreted. UniProt, BenignNodules, Literature, SymptomsDetection, Prediction CMGA_HUMAN Chromogranin-A CHGA LungCancers,Secreted. UniProt, BenignNodules Note = Neuro- Literature, endocrine andDetection, endocrine Prediction secretory granules. CNTN1_HUMANContactin-1 CNTN1 LungCancers Isoform 1: Cell Detection, membrane;Prediction Lipid-anchor, GPI- anchor; Extracellular side.|Isoform 2:Cell membrane; Lipid-anchor, GPI- anchor; Extracellular side.CO4A1_HUMAN Collagen COL4A1 LungCancers Secreted, UniProt, alpha-1(IV)extracellular Detection, chain space, Prediction extracellular matrix,basement membrane. CO5A2_HUMAN Collagen COL5A2 LungCancers Secreted,UniProt, alpha-2(V) extracellular Detection, chain space, Predictionextracellular matrix (By similarity). CO6A3_HUMAN Collagen COL6A3Secreted Symptoms Secreted, UniProt, alpha-3(VI) extracellularDetection, chain space, Prediction extracellular matrix (By similarity).COCA1_HUMAN Collagen COL12A1 ENDO LungCancers, Secreted, UniProt,alpha-1(XII) Symptoms extracellular Prediction chain space,extracellular matrix (By similarity). COF1_HUMAN Cofilin-1 CFL1Secreted, LungCancers, Nucleus matrix. Detection, EPI BenignNodulesCytoplasm, Prediction cytoskeleton. Note = Almost completely in nucleusin cells exposed to heat shock or 10% dimethyl sulfoxide. COIA1_HUMANCollagen COL18A1 LungCancers, Secreted, UniProt, alpha-1(XVIII)BenignNodules extracellular Literature, chain space, Detection,extracellular Prediction matrix (By similarity). COX5A_HUMAN CytochromeCOX5A Secreted, Mitochondrion Prediction c oxidase ENDO inner subunit5A, membrane. mitochondrial CRP_HUMAN C-reactive CRP LungCancers,Secreted. UniProt, protein BenignNodules, Literature, SymptomsDetection, Prediction CS051_HUMAN UPF0470 C19orf51 ENDO Predictionprotein C19orf51 CSF1_HUMAN Macrophage CSF1 LungCancers, Cell membrane;UniProt, colony- BenignNodules Single-pass Literature, stimulatingmembrane Detection factor 1 protein (By similarity).|Processedmacrophage colony- stimulating factor 1: Secreted, extracellular space(By similarity). CSF2_HUMAN Granulocyte- CSF2 LungCancers, Secreted.UniProt, macrophage BenignNodules Literature, colony- Predictionstimulating factor CT085_HUMAN Uncharacterized C20orf85 LungCancers,Prediction protein BenignNodules C20orf85 CTGF_HUMAN Connective CTGFLungCancers, Secreted, UniProt, tissue BenignNodules extracellularLiterature, growth space, Detection, factor extracellular Predictionmatrix (By similarity). Secreted (By similarity). CYR61_HUMAN ProteinCYR61 LungCancers, Secreted. UniProt, CYR61 BenignNodules PredictionCYTA_HUMAN Cystatin-A CSTA LungCancers Cytoplasm. Literature, DetectionCYTB_HUMAN Cystatin-B CSTB Secreted Cytoplasm. Literature, Nucleus.Detection DDX17_HUMAN Probable DDX17 ENDO LungCancers, Nucleus.Detection, ATP- BenignNodules Prediction dependent RNA helicase DDX17DEFB1_HUMAN Beta- DEFB1 LungCancers, Secreted. UniProt, defensin 1BenignNodules Prediction DESP_HUMAN Desmoplakin DSP EPI, ENDOLungCancers Cell junction, Detection desmosome. Cytoplasm, cytoskeleton.Note = Innermost portion of the desmosomal plaque. DFB4A_HUMAN Beta-DEFB4A LungCancers, Secreted. UniProt defensin 4A BenignNodulesDHI1L_HUMAN Hydroxysteroid HSD11B1L LungCancers Secreted UniProt,11-beta- (Potential). Prediction dehydrogenase 1-like proteinDMBT1_HUMAN Deleted in DMBT1 LungCancers, Secreted (By UniProt,malignant BenignNodules similarity). Detection, brain Note = SomePrediction tumors 1 isoforms may protein be membrane- bound. Localizedto the lumenal aspect of crypt cells in the small intestine. In thecolon, seen in the lumenal aspect of surface epithelial cells. Formed inthe ducts of von Ebner gland, and released into the fluid bathing thetaste buds contained in the taste papillae (By similarity). DMKN_HUMANDermokine DMKN LungCancers Secreted. UniProt, Detection, PredictionDPP4_HUMAN Dipeptidyl DPP4 EPI LungCancers, Dipeptidyl UniProt,peptidase 4 BenignNodules, peptidase 4 Detection Symptoms soluble form:Secreted.|Cell membrane; Single-pass type II membrane protein.DSG2_HUMAN Desmoglein-2 DSG2 ENDO Symptoms Cell membrane; UniProt,Single-pass Detection type I membrane protein. Cell junction, desmosome.DX39A_HUMAN ATP- DDX39A EPI Nucleus (By Prediction dependentsimilarity). RNA helicase DDX39A DX39B_HUMAN Spliceosome DDX39B EPINucleus. Prediction RNA Nucleus helicase speckle. DDX39B DYRK2_HUMANDual DYRK2 ENDO LungCancers Cytoplasm. Literature specificity Nucleus.tyrosine- Note = Translocates phosphorylation- into the regulatednucleus kinase 2 following DNA damage. EDN2_HUMAN Endothelin-2 EDN2LungCancers Secreted. UniProt, Prediction EF1A1_HUMAN Elongation EEF1A1Secreted, LungCancers, Cytoplasm. Detection factor 1- EPI BenignNodulesalpha 1 EF1D_HUMAN Elongation EEF1D Secreted, LungCancers Predictionfactor 1- EPI delta EF2_HUMAN Elongation EEF2 Secreted, Cytoplasm.Literature, factor 2 EPI Detection EGF_HUMAN Pro- EGF LungCancers,Membrane; UniProt, epidermal BenignNodules, Single-pass Literaturegrowth Symptoms type I factor membrane protein. EGFL6_HUMAN EpidermalEGFL6 LungCancers Secreted, UniProt, growth extracellular Detection,factor-like space, Prediction protein 6 extracellular matrix, basementmembrane (By similarity). ENOA_HUMAN Alpha- ENO1 Secreted, LungCancers,Cytoplasm. Cell Literature, enolase EPI, ENDO BenignNodules, membrane.Detection, Symptoms Cytoplasm, Prediction myofibril, sarcomere, M- band.Note = Can translocate to the plasma membrane in either the homodimeric(alpha/alpha) or heterodimeric (alpha/gamma) form. ENO1 is localized tothe M-band.|Isoform MBP-1: Nucleus. ENOG_HUMAN Gamma- ENO2 EPILungCancers, Cytoplasm (By Literature, enolase Symptoms similarity).Cell Detection, membrane (By Prediction similarity). Note = Cantranslocate to the plasma membrane in either the homodimeric(alpha/alpha) or heterodimeric (alpha/gamma) form (By similarity).ENOX2_HUMAN Ecto-NOX ENOX2 LungCancers Cell membrane. UniProt,disulfide- Secreted, Detection thiol extracellular exchanger space. 2Note = Extracellular and plasma membrane- associated. ENPL_HUMANEndoplasmin HSP90B1 Secreted, LungCancers, Endoplasmic Literature, EPI,ENDO BenignNodules, reticulum Detection, Symptoms lumen. PredictionMelanosome. Note = Identified by mass spectrometry in melanosomefractions from stage I to stage IV. EPHB6_HUMAN Ephrin EPHB6 LungCancersMembrane; UniProt, type-B Single-pass Literature receptor 6 type Imembrane protein.|Isoform 3: Secreted (Probable). EPOR_HUMANErythropoietin EPOR LungCancers, Cell membrane; UniProt, receptorBenignNodules, Single-pass Literature, Symptoms type I Detectionmembrane protein.|Isoform EPOR-S: Secreted. Note = Secreted and locatedto the cell surface. ERBB3_HUMAN Receptor ERBB3 LungCancers, Isoform 1:Cell UniProt, tyrosine- BenignNodules membrane; Literature, proteinSingle-pass Prediction kinase type I erbB-3 membrane protein.|Isoform 2:Secreted. EREG_HUMAN Proepiregulin EREG LungCancers Epiregulin: UniProtSecreted, extracellular space. Proepiregulin: Cell membrane; Single-passtype I membrane protein. ERO1A_HUMAN ERO1-like ERO1L Secreted, SymptomsEndoplasmic Prediction protein EPI, ENDO reticulum alpha membrane;Peripheral membrane protein; Lumenal side. Note = The association withERP44 is essential for its retention in the endoplasmic reticulum.ESM1_HUMAN Endothelial ESM1 LungCancers, Secreted. UniProt, cell-BenignNodules Prediction specific molecule 1 EZRI_HUMAN Ezrin EZRSecreted LungCancers, Apical cell Literature, BenignNodules membrane;Detection, Peripheral Prediction membrane protein; Cytoplasmic side.Cell projection. Cell projection, microvillus membrane; Peripheralmembrane protein; Cytoplasmic side. Cell projection, ruffle membrane;Peripheral membrane protein; Cytoplasmic side. Cytoplasm, cell cortex.Cytoplasm, cytoskeleton. Note = Localization to the apical membrane ofparietal cells depends on the interaction with MPP5. Localizes to cellextensions and peripheral processes of astrocytes (By similarity).Microvillar peripheral membrane protein (cytoplasmic side). F10A1_HUMANHsc70- ST13 EPI Cytoplasm (By Detection, interactingsimilarity).|Cytoplasm Prediction protein (Probable). FAM3C_HUMANProtein FAM3C EPI, ENDO Secreted UniProt, FAM3C (Potential). DetectionFAS_HUMAN Fatty acid FASN EPI LungCancers, Cytoplasm. Literature,synthase BenignNodules, Melanosome. Detection Symptoms Note = Identifiedby mass spectrometry in melanosome fractions from stage I to stage IV.FCGR1_HUMAN High FCGR1A EPI LungCancers, Cell membrane; UniProt affinityBenignNodules, Single-pass immunoglobulin Symptoms type I gamma Fcmembrane receptor I protein. Note = Stabilized at the cell membranethrough interaction with FCER1G. FGF10_HUMAN Fibroblast FGF10LungCancers Secreted UniProt, growth (Potential). Prediction factor 10FGF2_HUMAN Heparin- FGF2 LungCancers, Literature binding BenignNodules,growth Symptoms factor 2 FGF7_HUMAN Keratinocyte FGF7 LungCancers,Secreted. UniProt, growth BenignNodules Literature, factor PredictionFGF9_HUMAN Glia- FGF9 LungCancers Secreted. UniProt, activatingLiterature, factor Prediction FGFR2_HUMAN Fibroblast FGFR2 LungCancers,Cell membrane; UniProt, growth BenignNodules Single-pass Literature,factor type I Prediction receptor 2 membrane protein.|Isoform 14:Secreted.|Isoform 19: Secreted. FGFR3_HUMAN Fibroblast FGFR3 LungCancersMembrane; UniProt, growth Single-pass Literature, factor type IPrediction receptor 3 membrane protein. FGL2_HUMAN Fibroleukin FGL2BenignNodules, Secreted. UniProt, Symptoms Detection, PredictionFHIT_HUMAN Bis(5′- FHIT LungCancers, Cytoplasm. Literature adenosyl)-BenignNodules, triphosphatase Symptoms FIBA_HUMAN Fibrinogen FGALungCancers, Secreted. UniProt, alpha chain BenignNodules, Literature,Symptoms Detection, Prediction FINC_HUMAN Fibronectin FN1 Secreted,LungCancers, Secreted, UniProt, EPI, ENDO BenignNodules, extracellularLiterature, Symptoms space, Detection, extracellular Prediction matrix.FKB11_HUMAN Peptidyl- FKBP11 EPI, ENDO Membrane; UniProt, prolyl cis-Single-pass Prediction trans membrane isomerase protein FKBP11(Potential). FOLH1_HUMAN Glutamate FOLH1 ENDO LungCancers, Cellmembrane; UniProt, carboxypeptidase Symptoms Single-pass Literature 2type II membrane protein.|Isoform PSMA′: Cytoplasm. FOLR1_HUMAN FolateFOLR1 LungCancers Cell membrane; UniProt receptor Lipid-anchor, alphaGPI-anchor. Secreted (Probable). FOXA2_HUMAN Hepatocyte FOXA2LungCancers Nucleus. Detection, nuclear Prediction factor 3- betaFP100_HUMAN Fanconi C17orf70 ENDO Symptoms Nucleus. Prediction anemia-associated protein of 100 kDa FRIH_HUMAN Ferritin FTH1 EPI LungCancers,Literature, heavy BenignNodules Detection, chain Prediction FRIL_HUMANFerritin FTL Secreted, BenignNodules, Literature, light chain EPI, ENDOSymptoms Detection G3P_HUMAN Glyceralde- GAPDH Secreted, LungCancers,Cytoplasm. Detection hyde-3- EPI, ENDO BenignNodules, Cytoplasm,phosphate Symptoms perinuclear dehydrogenase region. Membrane. Note =Postnuclear and Perinuclear regions. G6PD_HUMAN Glucose-6- G6PDSecreted, LungCancers, Literature, phosphate EPI Symptoms Detection 1-dehydrogenase G6PI_HUMAN Glucose-6- GPI Secreted, Symptoms Cytoplasm.UniProt, phosphate EPI Secreted. Literature, isomerase DetectionGA2L1_HUMAN GAS2-like GAS2L1 ENDO Cytoplasm, Prediction protein 1cytoskeleton (Probable). GALT2_HUMAN Polypeptide N- GALNT2 EPI, ENDOGolgi UniProt, acetylgalactosaminyl- apparatus, Detection transferase 2Golgi stack membrane; Single-pass type II membrane protein. Secreted.Note = Resides preferentially in the trans and medial parts of the Golgistack. A secreted form also exists. GAS6_HUMAN Growth GAS6 LungCancersSecreted. UniProt, arrest- Detection, specific Prediction protein 6GDIR2_HUMAN Rho GDP- ARHGDIB EPI Cytoplasm. Detection dissociationinhibitor 2 GELS_HUMAN Gelsolin GSN LungCancers, Isoform 2: UniProt,BenignNodules Cytoplasm, Literature, cytoskeleton.|Isoform Detection, 1:Secreted. Prediction GGH_HUMAN Gamma- GGH LungCancers Secreted, UniProt,glutamyl extracellular Detection, hydrolase space. Prediction Lysosome.Melanosome. Note = While its intracellular location is primarily thelysosome, most of the enzyme activity is secreted. Identified by massspectrometry in melanosome fractions from stage I to stage IV.GPC3_HUMAN Glypican-3 GPC3 LungCancers, Cell membrane; UniProt, SymptomsLipid-anchor, Literature, GPI-anchor; Prediction Extracellular side (Bysimilarity).|Secreted glypican-3: Secreted, extracellular space (Bysimilarity). GRAN_HUMAN Grancalcin GCA EPI Cytoplasm. PredictionCytoplasmic granule membrane; Peripheral membrane protein; Cytoplasmicside. Note = Primarily cytosolic in the absence of calcium or magnesiumions. Relocates to granules and other membranes in response to elevatedcalcium and magnesium levels. GREB1_HUMAN Protein GREB1 ENDO Membrane;UniProt, GREB1 Single-pass Prediction membrane protein (Potential).GREM1_HUMAN Gremlin-1 GREM1 LungCancers, Secreted UniProt, BenignNodules(Probable). Prediction GRP_HUMAN Gastrin- GRP LungCancers, Secreted.UniProt, releasing Symptoms Prediction peptide GRP78_HUMAN 78 kDa HSPA5Secreted, LungCancers, Endoplasmic Detection, glucose- EPI, ENDOBenignNodules reticulum Prediction regulated lumen. protein Melanosome.Note = Identified by mass spectrometry in melanosome fractions fromstage I to stage IV. GSLG1_HUMAN Golgi GLG1 EPI, ENDO BenignNodulesGolgi apparatus UniProt apparatus membrane; protein 1 Single-pass type Imembrane protein. GSTP1_HUMAN Glutathione GSTP1 Secreted LungCancers,Literature, S- BenignNodules, Detection, transferase Symptoms PredictionP GTR1_HUMAN Solute SLC2A1 EPI, ENDO LungCancers, Cell membrane;Literature carrier BenignNodules, Multi-pass family 2, Symptoms membranefacilitated protein (By glucose similarity). transporter Melanosome.member 1 Note = Localizes primarily at the cell surface (By similarity).Identified by mass spectrometry in melanosome fractions from stage I tostage IV. GTR3_HUMAN Solute SLC2A3 EPI Membrane; Detection carrierMulti-pass family 2, membrane facilitated protein. glucose transportermember 3 H2A1_HUMAN Histone HIST1H2AG Secreted Nucleus. Detection, H2Atype 1 Prediction H2A1B_HUMAN Histone HIST1H2AB Secreted Nucleus.Detection, H2A type Prediction 1-B/E H2A1C_HUMAN Histone HIST1H2ACSecreted Nucleus. Literature, H2A type Detection, 1-C PredictionH2A1D_HUMAN Histone HIST1H2AD Secreted Nucleus. Detection, H2A typePrediction 1-D HG2A_HUMAN HLA class CD74 LungCancers, Membrane; UniProt,II BenignNodules, Single-pass Literature histocompatibility Symptomstype II antigen membrane gamma protein chain (Potential). HGF_HUMANHepatocyte HGF LungCancers, Literature, growth BenignNodules, Predictionfactor Symptoms HMGA1_HUMAN High HMGA1 LungCancers, Nucleus. Literaturemobility BenignNodules, group Symptoms protein HMG- I/HMG-Y HPRT_HUMANHypoxanthine- HPRT1 EPI Cytoplasm. Detection, guanine Predictionphosphoribosyl- transferase HPSE_HUMAN Heparanase HPSE LungCancers,Lysosome UniProt, BenignNodules, membrane; Prediction SymptomsPeripheral membrane protein. Secreted. Note = Secreted, internalised andtransferred to late endosomes/ lysosomes as a proheparanase. Inlysosomes, it is processed into the active form, the heparanase. Theuptake or internalisation of proheparanase is mediated by HSPGs. Heparinappears to be a competitor and retain proheparanase in the extracellularmedium. HPT_HUMAN Haptoglobin HP LungCancers, Secreted. UniProt,BenignNodules, Literature, Symptoms Detection, Prediction HS90A_HUMANHeat shock HSP90AA1 Secreted, LungCancers, Cytoplasm. Literature,protein EPI Symptoms Melanosome. Detection HSP 90- Note = Identifiedalpha by mass spectrometry in melanosome fractions from stage I to stageIV. HS90B_HUMAN Heat shock HSP90AB1 Secreted, LungCancers Cytoplasm.Literature, protein EPI Melanosome. Detection HSP 90- Note = Identifiedbeta by mass spectrometry in melanosome fractions from stage I to stageIV. HSPB1_HUMAN Heat shock HSPB1 Secreted, LungCancers, Cytoplasm.Literature, protein EPI BenignNodules Nucleus. Detection, beta-1Cytoplasm, Prediction cytoskeleton, spindle. Note = Cytoplasmic ininterphase cells. Colocalizes with mitotic spindles in mitotic cells.Translocates to the nucleus during heat shock. HTRA1_HUMAN Serine HTRA1LungCancers Secreted. UniProt, protease Prediction HTRA1 HXK1_HUMANHexokinase-1 HK1 ENDO Symptoms Mitochondrion Literature, outer Detectionmembrane. Note = Its hydrophobic N- terminal sequence may be involved inmembrane binding. HYAL2_HUMAN Hyaluronidase-2 HYAL2 LungCancers Cellmembrane; Prediction Lipid-anchor, GPI-anchor. HYOU1_HUMAN Hypoxia HYOU1EPI, ENDO Symptoms Endoplasmic Detection up- reticulum regulated lumen.protein 1 IBP2_HUMAN Insulin-like IGFBP2 LungCancers Secreted. UniProt,growth Literature, factor- Detection, binding Prediction protein 2IBP3_HUMAN Insulin-like IGFBP3 LungCancers, Secreted. UniProt, growthBenignNodules, Literature, factor- Symptoms Detection, bindingPrediction protein 3 ICAM1_HUMAN Intercellular ICAM1 LungCancers,Membrane; UniProt, adhesion BenignNodules, Single-pass Literature,molecule 1 Symptoms type I Detection membrane protein. ICAM3_HUMANIntercellular ICAM3 EPI, ENDO LungCancers, Membrane; UniProt, adhesionBenignNodules, Single-pass Detection molecule 3 Symptoms type I membraneprotein. IDHP_HUMAN Isocitrate IDH2 Secreted, Mitochondrion. Predictiondehydrogenase ENDO [NADP], mitochondrial IF4A1_HUMAN Eukaryotic EIF4A1Secreted, Detection, initiation EPI, ENDO Prediction factor 4A-IIGF1_HUMAN Insulin-like IGF1 LungCancers, Secreted.|Secreted. UniProt,growth BenignNodules, Literature, factor I Symptoms Detection,Prediction IKIP_HUMAN Inhibitor of IKIP ENDO Symptoms EndoplasmicUniProt, nuclear reticulum Prediction factor membrane; kappa-BSingle-pass kinase- membrane interacting protein. protein Note = Isoform4 deletion of the hydrophobic, or transmembrane region between AA 45-63results in uniform distribution troughout the cell, suggesting that thisregion is responsible for endoplasmic reticulum localization. IL18_HUMANInterleukin-18 IL18 LungCancers, Secreted. UniProt, BenignNodules,Literature, Symptoms Prediction IL19_HUMAN Interleukin-19 IL19LungCancers Secreted. UniProt, Detection, Prediction IL22_HUMANInterleukin-22 IL22 LungCancers, Secreted. UniProt, BenignNodulesPrediction IL32_HUMAN Interleukin-32 IL32 LungCancers, Secreted.UniProt, BenignNodules Prediction IL7_HUMAN Interleukin-7 IL7LungCancers, Secreted. UniProt, BenignNodules Literature, PredictionIL8_HUMAN Interleukin-8 IL8 LungCancers, Secreted. UniProt,BenignNodules, Literature Symptoms ILEU_HUMAN Leukocyte SERPINB1Secreted, Cytoplasm (By Detection, elastase EPI similarity). Predictioninhibitor ILK_HUMAN Integrin- ILK Secreted LungCancers, Cell junction,Literature, linked BenignNodules, focal adhesion. Detection proteinSymptoms Cell membrane; kinase Peripheral membrane protein; Cytoplasmicside. INHBA_HUMAN Inhibin INHBA LungCancers, Secreted. UniProt, beta ABenignNodules Literature, chain Prediction ISLR_HUMAN ImmunoglobulinISLR LungCancers Secreted UniProt, superfamily (Potential). Detection,containing Prediction leucine- rich repeat protein ITA5_HUMAN IntegrinITGA5 EPI LungCancers, Membrane; UniProt, alpha-5 BenignNodules,Single-pass Literature, Symptoms type I Detection membrane protein.ITAM_HUMAN Integrin ITGAM EPI, ENDO LungCancers, Membrane; UniProt,alpha-M BenignNodules, Single-pass Literature Symptoms type I membraneprotein. K0090_HUMAN Uncharacterized KIAA0090 EPI Symptoms Membrane;UniProt, protein Single-pass Prediction KIAA0090 type I membrane protein(Potential). K1C18_HUMAN Keratin, KRT18 Secreted LungCancers, Cytoplasm,Literature, type I BenignNodules perinuclear Detection, cytoskeletal 18region. Prediction K1C19_HUMAN Keratin, KRT19 LungCancers, Literature,type I BenignNodules Detection, cytoskeletal 19 Prediction K2C8_HUMANKeratin, KRT8 EPI LungCancers Cytoplasm. Literature, type II Detectioncytoskeletal 8 KIT_HUMAN Mast/stem KIT LungCancers Membrane; UniProt,cell growth Single-pass Literature, factor type I Detection receptormembrane protein. KITH_HUMAN Thymidine TK1 LungCancers Cytoplasm.Literature, kinase, Prediction cytosolic KLK11_HUMAN Kallikrein-11 KLK11LungCancers Secreted. UniProt, Literature, Prediction KLK13_HUMANKallikrein-13 KLK13 LungCancers Secreted UniProt, (Probable).Literature, Detection, Prediction KLK14_HUMAN Kallikrein-14 KLK14LungCancers, Secreted, UniProt, Symptoms extracellular Literature,space. Prediction KLK6_HUMAN Kallikrein-6 KLK6 LungCancers, Secreted.UniProt, BenignNodules, Nucleus, Literature, Symptoms nucleolus.Detection, Cytoplasm. Prediction Mitochondrion. Microsome. Note = Inbrain, detected in the nucleus of glial cells and in the nucleus andcytoplasm of neurons. Detected in the mitochondrial and microsomalfractions of HEK-293 cells and released into the cytoplasm followingcell stress. KNG1_HUMAN Kininogen-1 KNG1 LungCancers, Secreted, UniProt,BenignNodules, extracellular Detection, Symptoms space. PredictionKPYM_HUMAN Pyruvate PKM2 Secreted, LungCancers, Cytoplasm. Literature,kinase EPI Symptoms Nucleus. Detection isozymes Note = TranslocatesM1/M2 to the nucleus in response to different apoptotic stimuli. Nucleartranslocation is sufficient to induce cell death that is caspaseindependent, isoform-specific and independent of its enzymatic actvity.KRT35_HUMAN Keratin, KRT35 ENDO Detection, type I Prediction cuticularHa5 LAMB2_HUMAN Laminin LAMB2 ENDO LungCancers, Secreted, UniProt,subunit Symptoms extracellular Detection, beta-2 space, Predictionextracellular matrix, basement membrane. Note = S-laminin isconcentrated in the synaptic cleft of the neuromuscular junction.LDHA_HUMAN L-lactate LDHA Secreted, LungCancers Cytoplasm. Literature,dehydrogenase EPI, ENDO Detection, A chain Prediction LDHB_HUMANL-lactate LDHB EPI LungCancers Cytoplasm. Detection, dehydrogenasePrediction B chain LEG1_HUMAN Galectin-1 LGALS1 Secreted LungCancersSecreted, UniProt, extracellular Detection space, extracellular matrix.LEG3_HUMAN Galectin-3 LGALS3 LungCancers, Nucleus. Literature,BenignNodules Note = Cytoplasmic Detection, in adenomas and Predictioncarcinomas. May be secreted by a non- classical secretory pathway andassociate with the cell surface. LEG9_HUMAN Galectin-9 LGALS9 ENDOSymptoms Cytoplasm (By UniProt similarity). Secreted (By similarity).Note = May also be secreted by a non-classical secretory pathway (Bysimilarity). LG3BP_HUMAN Galectin-3- LGALS3BP Secreted LungCancers,Secreted. UniProt, binding BenignNodules, Secreted, Literature, proteinSymptoms extracellular Detection, space, Prediction extracellularmatrix. LPLC3_HUMAN Long C20orf185 LungCancers Secreted (By UniProt,palate, lung similarity). Prediction and nasal Cytoplasm. epitheliumNote = According to carcinoma- PubMed: 12837268 associated it iscytoplasmic. protein 3 LPLC4_HUMAN Long C20orf186 LungCancers Secreted(By UniProt, palate, lung similarity). Prediction and nasal Cytoplasm.epithelium carcinoma- associated protein 4 LPPRC_HUMAN Leucine- LRPPRCSecreted, LungCancers, Mitochondrion. Prediction rich PPR ENDO SymptomsNucleus, motif- nucleoplasm. containing Nucleus inner protein, membrane.mitochondrial Nucleus outer membrane. Note = Seems to be predominantlymitochondrial. LRP1_HUMAN Prolow- LRP1 EPI LungCancers, Low-densityUniProt, density Symptoms lipoprotein Detection lipoproteinreceptor-related receptor- protein 1 85 related kDa subunit: protein 1Cell membrane; Single-pass type I membrane protein. Membrane, coatedpit.|Low- density lipoprotein receptor-related protein 1 515 kDasubunit: Cell membrane; Peripheral membrane protein; Extracellular side.Membrane, coated pit.|Low- density lipoprotein receptor-related protein1 intracellular domain: Cytoplasm. Nucleus. Note = After cleavage, theintracellular domain (LRPICD) is detected both in the cytoplasm and inthe nucleus. LUM_HUMAN Lumican LUM Secreted, LungCancers, Secreted,UniProt, EPI BenignNodules, extracellular Detection, Symptoms space,Prediction extracellular matrix (By similarity). LY6K_HUMAN LymphocyteLY6K LungCancers, Secreted. UniProt, antigen Symptoms Cytoplasm. CellPrediction 6K membrane; Lipid- anchor, GPI-anchor (Potential).LYAM2_HUMAN E-selectin SELE LungCancers, Membrane; UniProt,BenignNodules, Single-pass Literature, Symptoms type I Detectionmembrane protein. LYAM3_HUMAN P-selectin SELP LungCancers, Membrane;UniProt, BenignNodules, Single-pass Literature, Symptoms type IDetection membrane protein. LYOX_HUMAN Protein- LOX LungCancers,Secreted, UniProt, lysine 6- BenignNodules extracellular Detection,oxidase space. Prediction LYPD3_HUMAN Ly6/PLAUR LYPD3 LungCancers Cellmembrane; Detection, domain- Lipid-anchor, Prediction containingGPI-anchor. protein 3 MAGA4_HUMAN Melanoma- MAGEA4 LungCancersLiterature, associated Prediction antigen 4 MASP1_HUMAN Mannan- MASP1LungCancers, Secreted. UniProt, binding Symptoms Detection, lectinPrediction serine protease 1 MDHC_HUMAN Malate MDH1 Secreted Cytoplasm.Literature, dehydrogenase, Detection, cytoplasmic Prediction MDHM_HUMANMalate MDH2 ENDO LungCancers Mitochondrion Detection, dehydrogenase,matrix. Prediction mitochondrial MIF_HUMAN Macrophage MIF SecretedLungCancers, Secreted. UniProt, migration BenignNodules, Cytoplasm.Literature, inhibitory Symptoms Note = Does not Prediction factor have acleavable signal sequence and is secreted via a specialized, non-classical pathway. Secreted by macrophages upon stimulation by bacteriallipopolysaccharide (LPS), or by M. tuberculosis antigens. MLH1_HUMAN DNAMLH1 ENDO LungCancers, Nucleus. Literature mismatch BenignNodules,repair Symptoms protein Mlh1 MMP1_HUMAN Interstitial MMP1 LungCancers,Secreted, UniProt, collagenase BenignNodules, extracellular Literature,Symptoms space, Prediction extracellular matrix (Probable). MMP11_HUMANStromelysin-3 MMP11 LungCancers, Secreted, UniProt, Symptomsextracellular Literature, space, Prediction extracellular matrix(Probable). MMP12_HUMAN Macrophage MMP12 LungCancers, Secreted, UniProt,metalloelastase BenignNodules, extracellular Literature, Symptoms space,Prediction extracellular matrix (Probable). MMP14_HUMAN Matrix MMP14ENDO LungCancers, Membrane; UniProt, metallopro- BenignNodules,Single-pass Literature, teinase-14 Symptoms type I Detection membraneprotein (Potential). Melanosome. Note = Identified by mass spectrometryin melanosome fractions from stage I to stage IV. MMP2_HUMAN 72 kDa MMP2LungCancers, Secreted, UniProt, type IV BenignNodules, extracellularLiterature, collagenase Symptoms space, Detection, extracellularPrediction matrix (Probable). MMP26_HUMAN Matrix MMP26 LungCancersSecreted, UniProt, metallopro- extracellular Prediction teinase-26space, extracellular matrix. MMP7_HUMAN Matrilysin MMP7 LungCancers,Secreted, UniProt, BenignNodules, extracellular Literature, Symptomsspace, Prediction extracellular matrix (Probable). MMP9_HUMAN MatrixMMP9 LungCancers, Secreted, UniProt, metallopro- BenignNodules,extracellular Literature, teinase-9 Symptoms space, Detection,extracellular Prediction matrix (Probable). MOGS_HUMAN Mannosyl- MOGSENDO Endoplasmic UniProt, oligosaccharide reticulum Predictionglucosidase membrane; Single-pass type II membrane protein. MPRI_HUMANCation- IGF2R EPI, ENDO LungCancers, Lysosome UniProt, independentSymptoms membrane; Literature, mannose-6- Single-pass Detectionphosphate type I receptor membrane protein. MRP3_HUMAN Canalicular ABCC3EPI LungCancers Membrane; Literature, multispecific Multi-pass Detectionorganic membrane anion protein. transporter 2 MUC1_HUMAN Mucin-1 MUC1EPI LungCancers, Apical cell UniProt, BenignNodules, membrane;Literature, Symptoms Single-pass Prediction type I membrane protein.Note = Exclusively located in the apical domain of the plasma membraneof highly polarized epithelial cells. After endocytosis, internalizedand recycled to the cell membrane. Located to microvilli and to the tipsof long filopodial protusions.|Isoform 5: Secreted.|Isoform 7:Secreted.|Isoform 9: Secreted.|Mucin-1 subunit beta: Cell membrane.Cytoplasm. Nucleus. Note = On EGF and PDGFRB stimulation, transported tothe nucleus through interaction with CTNNB1, a process which isstimulated by phosphorylation. On HRG stimulation, colocalizes withJUP/gamma- catenin at the nucleus. MUC16_HUMAN Mucin-16 MUC16LungCancers Cell membrane; UniProt, Single-pass Detection type Imembrane protein. Secreted, extracellular space. Note = May be liberatedinto the extracellular space following the phosphorylation of theintracellular C- terminus which induces the proteolytic cleavage andliberation of the extracellular domain. MUC4_HUMAN Mucin-4 MUC4LungCancers, Membrane; UniProt BenignNodules Single-pass membraneprotein (Potential). Secreted. Note = Isoforms lacking the Cys- richregion, EGF-like domains and transmembrane region are secreted.Secretion occurs by splicing or proteolytic processing. Mucin- 4 betachain: Cell membrane; Single- pass membrane protein.|Mucin- 4 alphachain: Secreted.|Isoform 3: Cell membrane; Single-pass membraneprotein.|Isoform 15: Secreted. MUC5B_HUMAN Mucin-5B MUC5B LungCancers,Secreted. UniProt, BenignNodules Detection, Prediction MUCL1_HUMANMucin-like MUCL1 LungCancers Secreted UniProt, protein 1 (Probable).Prediction Membrane (Probable). NAMPT_HUMAN Nicotinamide NAMPT EPILungCancers, Cytoplasm (By Literature, phosphoribosyl- BenignNodules,similarity). Detection transferase Symptoms NAPSA_HUMAN Napsin-A NAPSASecreted LungCancers Prediction NCF4_HUMAN Neutrophil NCF4 ENDOCytoplasm. Prediction cytosol factor 4 NDKA_HUMAN Nucleoside NME1Secreted LungCancers, Cytoplasm. Literature, diphosphate BenignNodules,Nucleus. Detection kinase A Symptoms Note = Cell- cycle dependentnuclear localization which can be induced by interaction withEpstein-barr viral proteins or by degradation of the SET complex byGzmA. NDKB_HUMAN Nucleoside NME2 Secreted, BenignNodules Cytoplasm.Literature, diphosphate EPI Nucleus. Detection kinase B Note = Isoform 2is mainly cytoplasmic and isoform 1 and isoform 2 are excluded from thenucleolus. NDUS1_HUMAN NADH- NDUFS1 Secreted, Symptoms MitochondrionPrediction ubiquinone ENDO inner oxidoreductase membrane. 75 kDasubunit, mitochondrial NEBL_HUMAN Nebulette NEBL ENDO PredictionNEK4_HUMAN Serine/threonine- NEK4 ENDO LungCancers Nucleus Predictionprotein (Probable). kinase Nek4 NET1_HUMAN Netrin-1 NTN1 LungCancers,Secreted, UniProt, BenignNodules extracellular Literature, space,Prediction extracellular matrix (By similarity). NEU2_HUMAN Vasopressin-AVP LungCancers, Secreted. UniProt, neurophysin 2- Symptoms Predictioncopeptin NGAL_HUMAN Neutrophil LCN2 EPI LungCancers, Secreted. UniProt,gelatinase- BenignNodules, Detection, associated Symptoms Predictionlipocalin NGLY1_HUMAN Peptide- NGLY1 ENDO Cytoplasm. Detection, N(4)-(N-Prediction acetyl-beta- glucosaminyl)as- paragine amidase NHRF1_HUMANNa(+)/H(+) SLC9A3R1 EPI BenignNodules Endomembrane Detection exchangesystem; regulatory Peripheral cofactor membrane NHE-RF1 protein. Cellprojection, filopodium. Cell projection, ruffle. Cell projection,microvillus. Note = Colocalizes with actin in microvilli-rich apicalregions of the syncytiotrophoblast. Found in microvilli, rufflingmembrane and filopodia of HeLa cells. Present in lipid rafts of T-cells.NIBAN_HUMAN Protein FAM129A EPI Cytoplasm. Literature, Niban DetectionNMU_HUMAN Neuromedin-U NMU LungCancers Secreted. UniProt, PredictionNRP1_HUMAN Neuropilin-1 NRP1 LungCancers, Cell membrane; UniProt,BenignNodules, Single-pass Literature, Symptoms type I Detection,membrane Prediction protein.|Isoform 2: Secreted. ODAM_HUMAN OdontogenicODAM LungCancers Secreted (By UniProt, ameloblast- similarity).Prediction associated protein OSTP_HUMAN Osteopontin SPP1 LungCancers,Secreted. UniProt, BenignNodules, Literature, Symptoms Detection,Prediction OVOS2_HUMAN Ovostatin OVOS2 ENDO Secreted (By UniProt,homolog 2 similarity). Prediction P5CS_HUMAN Delta-1- ALDH18A1 ENDOMitochondrion Prediction pyrroline-5- inner carboxylate membrane.synthase PA2GX_HUMAN Group 10 PLA2G10 Symptoms Secreted. UniProtsecretory phospholipase A2 PAPP1_HUMAN Pappalysin-1 PAPPA LungCancers,Secreted. UniProt, BenignNodules, Literature, Symptoms PredictionPBIP1_HUMAN Pre-B-cell PBXIP1 EPI Cytoplasm, Prediction leukemiacytoskeleton. transcription Nucleus. factor- Note = Shuttles interactingbetween the protein 1 nucleus and the cytosol. Mainly localized in thecytoplasm, associated with microtubules. Detected in small amounts inthe nucleus. PCBP1_HUMAN Poly(rC)- PCBP1 EPI, ENDO Nucleus. Detection,binding Cytoplasm. Prediction protein 1 Note = Loosely bound in thenucleus. May shuttle between the nucleus and the cytoplasm. PCBP2_HUMANPoly(rC)- PCBP2 EPI Nucleus. Detection, binding Cytoplasm. Predictionprotein 2 Note = Loosely bound in the nucleus. May shuttle between thenucleus and the cytoplasm. PCD15_HUMAN Protocadherin- PCDH15 ENDO Cellmembrane; UniProt, 15 Single-pass Detection type I membrane protein (Bysimilarity).|Isoform 3: Secreted. PCNA_HUMAN Proliferating PCNA EPILungCancers, Nucleus. Literature, cell nuclear BenignNodules, Predictionantigen Symptoms PCYOX_HUMAN Prenylcysteine PCYOX1 Secreted LungCancers,Lysosome. Detection, oxidase 1 Symptoms Prediction PDGFA_HUMAN Platelet-PDGFA LungCancers Secreted. UniProt, derived Literature, growthPrediction factor subunit A PDGFB_HUMAN Platelet- PDGFB LungCancers,Secreted. UniProt, derived BenignNodules, Literature, growth SymptomsDetection, factor Prediction subunit B PDGFD_HUMAN Platelet- PDGFDLungCancers Secreted. UniProt, derived Prediction growth factor DPDIA3_HUMAN Protein PDIA3 ENDO LungCancers Endoplasmic Detection,disulfide- reticulum Prediction isomerase lumen (By A3 similarity).Melanosome. Note = Identified by mass spectrometry in melanosomefractions from stage I to stage IV. PDIA4_HUMAN Protein PDIA4 Secreted,Endoplasmic Detection, disulfide- EPI, ENDO reticulum Predictionisomerase lumen. A4 Melanosome. Note = Identified by mass spectrometryin melanosome fractions from stage I to stage IV. PDIA6_HUMAN ProteinPDIA6 Secreted, Endoplasmic Detection, disulfide- EPI, ENDO reticulumPrediction isomerase lumen (By A6 similarity). Melanosome. Note =Identified by mass spectrometry in melanosome fractions from stage I tostage IV. PECA1_HUMAN Platelet PECAM1 LungCancers, Membrane; UniProt,endothelial BenignNodules, Single-pass Literature, cell Symptoms type IDetection adhesion membrane molecule protein. PEDF_HUMAN PigmentSERPINF1 LungCancers, Secreted. UniProt, epithelium- SymptomsMelanosome. Literature, derived Note = Enriched Detection, factor instage I Prediction melanosomes. PERM_HUMAN Myeloperoxidase MPO Secreted,LungCancers, Lysosome. Literature, EPI, ENDO BenignNodules, Detection,Symptoms Prediction PERP1_HUMAN Plasma PACAP EPI, ENDO Secreted UniProt,cell- (Potential). Detection, induced Cytoplasm. Prediction residentNote = In endoplasmic (PubMed: 11350957) reticulum diffuse granularprotein localization in the cytoplasm surrounding the nucleus.PGAM1_HUMAN Phosphoglycerate PGAM1 Secreted, LungCancers, Detectionmutase 1 EPI Symptoms PLAC1_HUMAN Placenta- PLAC1 LungCancers SecretedUniProt, specific (Probable). Prediction protein 1 PLACL_HUMAN Placenta-PLAC1L LungCancers Secreted UniProt, specific 1- (Potential). Predictionlike protein PLIN2_HUMAN Perilipin-2 ADFP ENDO LungCancers Membrane;Prediction Peripheral membrane protein. PLIN3_HUMAN Perilipin-3 M6PRBP1EPI Cytoplasm. Detection, Endosome Prediction membrane; Peripheralmembrane protein; Cytoplasmic side (Potential). Lipid droplet(Potential). Note = Membrane associated on endosomes. Detected in theenvelope and the core of lipid bodies and in lipid sails. PLOD1_HUMANProcollagen- PLOD1 EPI, ENDO Rough Prediction lysine, 2- endoplasmicoxoglutarate 5- reticulum dioxygenase 1 membrane; Peripheral membraneprotein; Lumenal side. PLOD2_HUMAN Procollagen- PLOD2 ENDOBenignNodules, Rough Prediction lysine, 2- Symptoms endoplasmicoxoglutarate 5- reticulum dioxygenase 2 membrane; Peripheral membraneprotein; Lumenal side. PLSL_HUMAN Plastin-2 LCP1 Secreted, LungCancersCytoplasm, Detection, EPI cytoskeleton. Prediction Cell junction. Cellprojection. Cell projection, ruffle membrane; Peripheral membraneprotein; Cytoplasmic side (By similarity). Note = Relocalizes to theimmunological synapse between peripheral blood T lymphocytes andantibody- presenting cells in response to costimulation through TCR/CD3and CD2 or CD28. Associated with the actin cytoskeleton at membraneruffles (By similarity). Relocalizes to actin-rich cell projections uponserine phosphorylation. PLUNC_HUMAN Protein PLUNC LungCancers, Secreted(By UniProt, Plunc BenignNodules similarity). Prediction Note = Found inthe nasal mucus (By similarity). Apical side of airway epithelial cells.Detected in nasal mucus (By similarity). PLXB3_HUMAN Plexin-B3 PLXNB3ENDO Membrane; UniProt, Single-pass Detection, type I Predictionmembrane protein. PLXC1_HUMAN Plexin-C1 PLXNC1 EPI Membrane; UniProt,Single-pass Detection type I membrane protein (Potential). POSTN_HUMANPeriostin POSTN Secreted, LungCancers, Secreted, UniProt, ENDOBenignNodules, extracellular Literature, Symptoms space, Detection,extracellular Prediction matrix. PPAL_HUMAN Lysosomal ACP2 EPI SymptomsLysosome UniProt, acid membrane; Prediction phosphatase Single-passmembrane protein; Lumenal side. Lysosome lumen. Note = The soluble formarises by proteolytic processing of the membrane- bound form. PPBT_HUMANAlkaline ALPL EPI LungCancers, Cell membrane; Literature, phosphatase,BenignNodules, Lipid-anchor, Detection, tissue- Symptoms GPI-anchor.Prediction nonspecific isozyme PPIB_HUMAN Peptidyl- PPIB Secreted,Endoplasmic Detection, prolyl cis- EPI, ENDO reticulum Prediction translumen. isomerase Melanosome. B Note = Identified by mass spectrometry inmelanosome fractions from stage I to stage IV. PRDX1_HUMANPeroxiredoxin- PRDX1 EPI LungCancers Cytoplasm. Detection, 1 Melanosome.Prediction Note = Identified by mass spectrometry in melanosomefractions from stage I to stage IV. PRDX4_HUMAN Peroxiredoxin- PRDX4Secreted, Cytoplasm. Literature, 4 EPI, ENDO Detection, PredictionPROF1_HUMAN Profilin-1 PFN1 Secreted, LungCancers Cytoplasm, DetectionEPI cytoskeleton. PRP31_HUMAN U4/U6 PRPF31 ENDO Nucleus Prediction smallspeckle. nuclear Nucleus, Cajal ribonucleo body. protein Note =Predominantly Prp31 found in speckles and in Cajal bodies. PRS6A_HUMAN26S PSMC3 EPI BenignNodules Cytoplasm Detection protease (Potential).regulatory Nucleus subunit 6A (Potential). PSCA_HUMAN Prostate PSCALungCancers Cell membrane; Literature, stem cell Lipid-anchor,Prediction antigen GPI-anchor. PTGIS_HUMAN Prostacyclin PTGIS EPILungCancers, Endoplasmic UniProt, synthase BenignNodules reticulumDetection, membrane; Prediction Single-pass membrane protein. PTPA_HUMANSerine/threonine- PPP2R4 ENDO Symptoms Detection, protein Predictionphosphatase 2A activator PTPRC_HUMAN Receptor- PTPRC Secreted,LungCancers Membrane; UniProt, type EPI, ENDO Single-pass Detection,tyrosine- type I Prediction protein membrane phosphatase C protein.PTPRJ_HUMAN Receptor- PTPRJ EPI LungCancers, Membrane; UniProt, typeSymptoms Single-pass Detection, tyrosine- type I Prediction proteinmembrane phosphatase eta protein. PVR_HUMAN Poliovirus PVR SymptomsIsoform Alpha: UniProt, receptor Cell membrane; Detection, Single-passPrediction type I membrane protein.|Isoform Delta: Cell membrane;Single-pass type I membrane protein.|Isoform Beta: Secreted.|IsoformGamma: Secreted. RAB32_HUMAN Ras-related RAB32 EPI Mitochondrion.Prediction protein Rab-32 RAGE_HUMAN Advanced AGER Secreted LungCancers,Isoform 1: Cell UniProt, glycosylation BenignNodules membrane;Literature end product- Single-pass specific type I receptor membraneprotein.|Isoform 2: Secreted. RAN_HUMAN GTP- RAN Secreted, LungCancers,Nucleus. Detection, binding EPI BenignNodules Cytoplasm. Predictionnuclear Melanosome. protein Ran Note = Becomes dispersed throughout thecytoplasm during mitosis. Identified by mass spectrometry in melanosomefractions from stage I to stage IV. RAP2B_HUMAN Ras-related RAP2B EPICell membrane; Prediction protein Lipid-anchor; Rap-2b Cytoplasmic side(Potential). RAP2C_HUMAN Ras-related RAP2C EPI Cell membrane; Predictionprotein Lipid-anchor; Rap-2c Cytoplasmic side (Potential). RCN3_HUMANReticulocalbin- RCN3 EPI Symptoms Endoplasmic Prediction 3 reticulumlumen (Potential). RL24_HUMAN 60S RPL24 EPI Prediction ribosomal proteinL24 S10A1_HUMAN Protein S100A1 Symptoms Cytoplasm. Literature, S100-A1Prediction S10A6_HUMAN Protein S100A6 Secreted LungCancers NucleusLiterature, S100-A6 envelope. Detection, Cytoplasm. PredictionS10A7_HUMAN Protein S100A7 LungCancers Cytoplasm. UniProt, S100-A7Secreted. Literature, Note = Secreted Detection, by a non- Predictionclassical secretory pathway. SAA_HUMAN Serum SAA1 Symptoms Secreted.UniProt, amyloid A Literature, protein Detection, Prediction SCF_HUMANKit ligand KITLG LungCancers, Isoform 1: Cell UniProt, Symptomsmembrane; Literature Single-pass type I membrane protein (Bysimilarity). Secreted (By similarity). Note = Also exists as a secretedsoluble form (isoform 1 only) (By similarity).|Isoform 2: Cell membrane;Single-pass type I membrane protein (By similarity). Cytoplasm,cytoskeleton (By similarity). SDC1_HUMAN Syndecan-1 SDC1 LungCancers,Membrane; UniProt, BenignNodules, Single-pass Literature, Symptoms typeI Detection membrane protein. SEM3G_HUMAN Semaphorin-3G SEMA3GLungCancers Secreted (By UniProt, similarity). Prediction SEPR_HUMANSeprase FAP ENDO Symptoms Cell membrane; UniProt, Single-passLiterature, type II Detection membrane protein. Cell projection,lamellipodium membrane; Single-pass type II membrane protein. Cellprojection, invadopodium membrane; Single-pass type II membrane protein.Note = Found in cell surface lamellipodia, invadopodia and on shedvesicles. SERPH_HUMAN Serpin H1 SERPINH1 Secreted, LungCancers,Endoplasmic Detection, EPI, ENDO BenignNodules reticulum Predictionlumen. SFPA2_HUMAN Pulmonary SFTPA2 Secreted LungCancers, Secreted,UniProt, surfactant- BenignNodules extracellular Prediction associatedspace, protein A2 extracellular matrix. Secreted, extracellular space,surface film. SFTA1_HUMAN Pulmonary SFTPA1 Secreted LungCancers,Secreted, UniProt, surfactant- BenignNodules, extracellular Predictionassociated Symptoms space, protein A1 extracellular matrix. Secreted,extracellular space, surface film. SG3A2_HUMAN Secretoglobin SCGB3A2LungCancers, Secreted. UniProt, family BenignNodules Prediction 3Amember 2 SGPL1_HUMAN Sphingosine-1- SGPL1 ENDO Endoplasmic UniProt,phosphate reticulum Prediction lyase 1 membrane; Single-pass type IIImembrane protein. SIAL_HUMAN Bone IBSP LungCancers Secreted. UniProt,sialoprotein Literature, 2 Prediction SLPI_HUMAN Antileukopro- SLPILungCancers, Secreted. UniProt, teinase BenignNodules Literature,Detection, Prediction SMD3_HUMAN Small SNRPD3 Secreted BenignNodulesNucleus. Prediction nuclear ribonucleo protein Sm D3 SMS_HUMANSomatostatin SST LungCancers Secreted. UniProt, Literature, PredictionSODM_HUMAN Superoxide SOD2 Secreted LungCancers, MitochondrionLiterature, dismutase BenignNodules, matrix. Detection, [Mn], SymptomsPrediction mitochondrial SORL_HUMAN Sortilin- SORL1 EPI LungCancers,Membrane; UniProt, related Symptoms Single-pass Detection receptor typeI membrane protein (Potential). SPB3_HUMAN Serpin B3 SERPINB3LungCancers, Cytoplasm. Literature, BenignNodules Note = Seems toDetection also be secreted in plasma by cancerous cells but at a lowlevel. SPB5_HUMAN Serpin B5 SERPINB5 LungCancers Secreted, UniProt,extracellular Detection space. SPON2_HUMAN Spondin-2 SPON2 LungCancers,Secreted, UniProt, BenignNodules extracellular Prediction space,extracellular matrix (By similarity). SPRC_HUMAN SPARC SPARCLungCancers, Secreted, UniProt, BenignNodules, extracellular Literature,Symptoms space, Detection, extracellular Prediction matrix, basementmembrane. Note = In or around the basement membrane. SRC_HUMAN Proto-SRC ENDO LungCancers, Literature oncogene BenignNodules, tyrosine-Symptoms protein kinase Src SSRD_HUMAN Translocon- SSR4 Secreted,Endoplasmic UniProt, associated ENDO reticulum Prediction proteinmembrane; subunit Single-pass delta type I membrane protein. STAT1_HUMANSignal STAT1 EPI LungCancers, Cytoplasm. Detection transducerBenignNodules Nucleus. and Note = Translocated activator of into thetranscription nucleus in 1-alpha/beta response to IFN-gamma- inducedtyrosine phosphorylation and dimerization. STAT3_HUMAN Signal STAT3 ENDOLungCancers, Cytoplasm. Prediction transducer BenignNodules, Nucleus.and Symptoms Note = Shuttles activator of between the transcriptionnucleus and the 3 cytoplasm. Constitutive nuclear presence isindependent of tyrosine phosphorylation. STC1_HUMAN Stanniocalcin- STC1LungCancers, Secreted. UniProt, 1 Symptoms Prediction STT3A_HUMANDolichyl- STT3A EPI Symptoms Endoplasmic Literature diphosphooligosac-reticulum charide--protein membrane; glycosyltransferase Multi-passsubunit membrane STT3A protein. TAGL_HUMAN Transgelin TAGLN EPILungCancers Cytoplasm Literature, (Probable). Prediction TARA_HUMAN TRIOand TRIOBP ENDO Nucleus. Detection, F-actin- Cytoplasm, Predictionbinding cytoskeleton. protein Note = Localized to F-actin in a periodicpattern. TBA1B_HUMAN Tubulin TUBA1B EPI LungCancers Detection alpha-1Bchain TBB2A_HUMAN Tubulin TUBB2A EPI LungCancers, Detection, beta-2ABenignNodules Prediction chain TBB3_HUMAN Tubulin TUBB3 EPI LungCancers,Detection beta-3 BenignNodules chain TBB5_HUMAN Tubulin TUBB EPILungCancers, Detection beta chain BenignNodules TCPA_HUMAN T-complexTCP1 EPI Cytoplasm. Prediction protein 1 subunit alpha TCPD_HUMANT-complex CCT4 EPI Cytoplasm. Detection, protein 1 Melanosome.Prediction subunit Note = Identified delta by mass spectrometry inmelanosome fractions from stage I to stage IV. TCPQ_HUMAN T-complex CCT8Secreted, Cytoplasm. Prediction protein 1 EPI subunit theta TCPZ_HUMANT-complex CCT6A Secreted, Cytoplasm. Detection protein 1 EPI subunitzeta TDRD3_HUMAN Tudor TDRD3 ENDO Cytoplasm. Prediction domain- Nucleus.containing Note = Predominantly protein 3 cytoplasmic. Associated withactively translating polyribosomes and with mRNA stress granules.TENA_HUMAN Tenascin TNC ENDO LungCancers, Secreted, UniProt,BenignNodules, extracellular Literature, Symptoms space, Detectionextracellular matrix. TENX_HUMAN Tenascin-X TNXB ENDO LungCancers,Secreted, UniProt, Symptoms extracellular Detection, space, Predictionextracellular matrix. TERA_HUMAN Transitional VCP EPI LungCancers,Cytoplasm, Detection endoplasmic BenignNodules cytosol. reticulumNucleus. ATPase Note = Present in the neuronal hyaline inclusion bodiesspecifically found in motor neurons from amyotrophic lateral sclerosispatients. Present in the Lewy bodies specifically found in neurons fromParkinson disease patients. TETN_HUMAN Tetranectin CLEC3B LungCancersSecreted. UniProt, Literature, Detection, Prediction TF_HUMAN Tissue F3LungCancers, Membrane; UniProt, factor BenignNodules, Single-passLiterature Symptoms type I membrane protein. TFR1_HUMAN Transferrin TFRCSecreted, LungCancers, Cell membrane; UniProt, receptor EPI, ENDOBenignNodules, Single-pass Literature, protein 1 Symptoms type IIDetection membrane protein. Melanosome. Note = Identified by massspectrometry in melanosome fractions from stage I to stageIV.|Transferrin receptor protein 1, serum form: Secreted. TGFA_HUMANProtransforming TGFA LungCancers, Transforming UniProt, growthBenignNodules growth factor Literature factor alpha alpha: Secreted,extracellular space.|Protransforming growth factor alpha: Cell membrane;Single-pass type I membrane protein. THAS_HUMAN Thromboxane- TBXAS1 EPI,ENDO LungCancers, Membrane; Prediction A BenignNodules, Multi-passsynthase Symptoms membrane protein. THY1_HUMAN Thy-1 THY1 EPI SymptomsCell membrane; Detection, membrane Lipid-anchor, Prediction glycoproteinGPI-anchor (By similarity). TIMP1_HUMAN Metalloproteinase TIMP1LungCancers, Secreted. UniProt, inhibitor 1 BenignNodules, Literature,Symptoms Detection, Prediction TIMP3_HUMAN Metalloproteinase TIMP3LungCancers, Secreted, UniProt, inhibitor 3 BenignNodules extracellularLiterature, space, Prediction extracellular matrix. TLL1_HUMAN Tolloid-TLL1 ENDO Secreted UniProt, like protein (Probable). Prediction 1TNF12_HUMAN Tumor TNFSF12 LungCancers, Cell membrane; UniProt necrosisBenignNodules Single-pass factor type II ligand membrane superfamilyprotein.|Tumor member 12 necrosis factor ligand superfamily member 12,secreted form: Secreted. TNR6_HUMAN Tumor FAS LungCancers, Isoform 1:Cell UniProt, necrosis BenignNodules, membrane; Literature, factorSymptoms Single-pass Prediction receptor type I superfamily membranemember 6 protein.|Isoform 2: Secreted.|Isoform 3: Secreted.|Isoform 4:Secreted.|Isoform 5: Secreted.|Isoform 6: Secreted. TPIS_HUMANTriosephosphate TPI1 Secreted, Symptoms Literature, isomerase EPIDetection, Prediction TRFL_HUMAN Lactotransferrin LTF Secreted,LungCancers, Secreted. UniProt, EPI, ENDO BenignNodules, Literature,Symptoms Detection, Prediction TSP1_HUMAN Thrombospondin- THBS1LungCancers, Literature, 1 BenignNodules, Detection, Symptoms PredictionTTHY_HUMAN Transthyretin TTR LungCancers, Secreted. UniProt,BenignNodules Cytoplasm. Literature, Detection, Prediction TYPH_HUMANThymidine TYMP EPI LungCancers, Literature, phosphorylase BenignNodules,Detection, Symptoms Prediction UGGG1_HUMAN UDP-glucose: UGGT1 Secreted,Endoplasmic Detection, glycoprotein ENDO reticulum Predictionglucosyltransferase lumen. 1 Endoplasmic reticulum-Golgi intermediatecompartment. UGGG2_HUMAN UDP-glucose: UGGT2 ENDO Endoplasmic Predictionglycoprotein reticulum glucosyltransferase lumen. 2 Endoplasmicreticulum-Golgi intermediate compartment. UGPA_HUMAN UTP-- UGP2 EPISymptoms Cytoplasm. Detection glucose-1- phosphate uridylyltransferaseUPAR_HUMAN Urokinase PLAUR LungCancers, Isoform 1: Cell UniProt,plasminogen BenignNodules, membrane; Literature, activator SymptomsLipid-anchor, Prediction surface GPI- receptor anchor.|Isoform 2:Secreted (Probable). UTER_HUMAN Uteroglobin SCGB1A1 LungCancers,Secreted. UniProt, BenignNodules, Literature, Symptoms Detection,Prediction VA0D1_HUMAN V-type ATP6V0D1 EPI Prediction proton ATPasesubunit d 1 VAV3_HUMAN Guanine VAV3 ENDO Prediction nucleotide exchangefactor VAV3 VEGFA_HUMAN Vascular VEGFA LungCancers, Secreted. UniProt,endothelial BenignNodules, Note = VEGF12 Literature, growth Symptoms 1is acidic and Prediction factor A freely secreted. VEGF165 is morebasic, has heparin-binding properties and, although a signicantproportion remains cell- associated, most is freely secreted. VEGF189 isvery basic, it is cell-associated after secretion and is bound avidly byheparin and the extracellular matrix, although it may be released as asoluble form by heparin, heparinase or plasmin. VEGFC_HUMAN VascularVEGFC LungCancers, Secreted. UniProt, endothelial BenignNodulesLiterature, growth Prediction factor C VEGFD_HUMAN Vascular FIGFLungCancers Secreted. UniProt, endothelial Literature, growth Predictionfactor D VGFR1_HUMAN Vascular FLT1 LungCancers, Isoform Flt1: UniProt,endothelial BenignNodules, Cell membrane; Literature, growth SymptomsSingle-pass Detection, factor type I Prediction receptor 1 membraneprotein.|Isoform sFlt1: Secreted. VTNC_HUMAN Vitronectin VTN ENDOSymptoms Secreted, UniProt, extracellular Literature, space. Detection,Prediction VWC2_HUMAN Brorin VWC2 LungCancers Secreted, UniProt,extracellular Prediction space, extracellular matrix, basement membrane(By similarity). WNT3A_HUMAN Protein WNT3A LungCancers, Secreted,UniProt, Wnt-3a Symptoms extracellular Prediction space, extracellularmatrix. WT1_HUMAN Wilms WT1 LungCancers, Nucleus. Literature, tumorBenignNodules, Cytoplasm (By Prediction protein Symptoms similarity).Note = Shuttles between nucleus and cytoplasm (By similarity).|Isoform1: Nucleus speckle.|Isoform 4: Nucleus, nucleoplasm. ZA2G_HUMANZinc-alpha- AZGP1 LungCancers, Secreted. UniProt, 2- SymptomsLiterature, glycoprotein Detection, Prediction ZG16B_HUMAN Zymogen ZG16BLungCancers Secreted UniProt, granule (Potential). Prediction protein 16homolog B

SRM Assay

SRM assays for 388 targeted proteins were developed based on syntheticpeptides, using a protocol similar to those described in the literature(Lange, Picotti et al. 2008, Picotti, Rinner et al. 2010, Huttenhain,Soste et al. 2012). Up to five SRM suitable peptides per protein wereidentified from public sources such as the PeptideAtlas, Human PlasmaProteome Database or by proteotypic prediction tools (Mallick, Schirleet al. 2007) and synthesized. SRM triggered MS/MS spectra were collectedon an ABSciex 5500 QTrap for both doubly and triply charged precursorions. The obtained MS/MS spectra were assigned to individual peptidesusing MASCOT (cutoff score≧15) (Perkins, Pappin et al. 1999). Up to fourtransitions per precursor ion were selected for optimization. Theresulting corresponding optimal retention time, declustering potentialand collision energy were assembled for all transitions. Optimaltransitions were measured on a mixture of all synthetic peptides, apooled sample of benign patients and a pooled sample of cancer patients.Transitions were analyzed in batches, each containing up to 1750transitions. Both biological samples were immuno-depleted and digestedby trypsin. All three samples were analyzed on an ABSciex 5500 QTrapcoupled with a reversed-phase (RP) high-performance liquidchromatography (HPLC) system. The obtained SRM data were manuallyreviewed to select the two best peptides per protein and the two besttransitions per peptide. Transitions having interference with othertransitions were not selected. Ratios between intensities of the twobest transitions of peptides in the synthetic peptide mixture were alsoused to assess the specificity of the transitions in the biologicalsamples. The intensity ratio was considered as an important metricdefining the SRM assays. The complete transition table is shown below inTable 2.

Lengthy table referenced here US20150323545A1-20151112-T00001 Pleaserefer to the end of the specification for access instructions.

Exemplary Protein Detection

The following 164 proteins and their peptides were detectedsimultaneously in a large-scale experiment of 158 samples using theMS-LC-SRM-MS system described herein.

TABLE 3 SEQ Protein Peptide ID Detec- Detec- Protein Peptide NO: tiontion 1433E_HUMAN EDLVYQAK 7 16 16 1433E_HUMAN IISSIEQK 9 16 01433T_HUMAN AVTEQGAELSNEER 16 127 0 1433T_HUMAN TAFDEAIAELDTLNED 19 127127 SYK 1433Z_HUMAN FLIPNASQAESK 21 157 157 1433Z_HUMAN SVTEQGAELSNEER23 157 0 6PGD_HUMAN AGQAVDDFIEK 25 90 0 6PGD_HUMAN LVPLLDTGDIIIDGGNS 2790 90 EYR A1AG1_HUMAN WFYIASAFR 32 157 0 A1AG1_HUMAN YVGGQEHFAHLLILR 33157 157 ABCD1_HUMAN DAGIALLSITHRPSLWK 34 9 0 ABCD1_HUMANGLQAPAGEPTQEASGVA 36 9 0 AAK ABCD1_HUMAN NLLTAAADAIER 37 9 9 ADML_HUMANLAHQIYQFTDK 44 27 27 ADML_HUMAN SPEDSSPDAAR 45 27 0 AIFM1_HUMANELWFSDDPNVTK 53 158 158 AIFM1_HUMAN GVIFYLR 54 158 0 ALDOA_HUMANADDGRPFPQVIK 57 158 141 ALDOA_HUMAN ALQASALK 58 158 17 AMPN_HUMANALEQALEK 62 158 158 AMPN_HUMAN DHSAIPVINR 63 158 0 APOA1_HUMAN AKPALEDLR78 158 158 APOA1_HUMAN ATEHLSTLSEK 79 158 0 APOE_HUMAN AATVGSLAGQPLQER82 158 158 APOE_HUMAN LGPLVEQGR 87 158 0 BGH3_HUMAN LTLLAPLNSVFK 139 1580 BGH3_HUMAN SPYQLVLQHSR 140 158 158 BST1_HUMAN GEGTSAHLR 149 157 0BST1_HUMAN GFFADYEIPNLQK 150 157 157 C163A_HUMAN INPASLDK 153 158 11C163A_HUMAN LEVFYNGAWGTVGK 154 158 49 C163A_HUMAN TSYQVYSK 155 158 98CALU_HUMAN EQFVEFR 172 120 120 CALU_HUMAN TFDQLTPEESK 174 120 0CATB_HUMAN LPASFDAR 188 62 62 CATB_HUMAN TDQYWEK 190 62 0 CATG_HUMANNVNPVALPR 192 14 0 CATG_HUMAN SSGVPPEVFTR 193 14 14 CBPB2_HUMANDTGTYGFLLPER 198 158 158 CBPB2_HUMAN EAFAAVSK 199 158 0 CD14_HUMANATVNPSAPR 207 158 0 CD14_HUMAN SWLAELQQWLKPGLK 214 158 158 CD44_HUMANFAGVFHVEK 227 158 158 CD44_HUMAN YGFIEGHVVIPR 231 158 0 CD59_HUMANAGLQVYNK 232 156 156 CD59_HUMAN TVLLLVTPFLAAAWSLHP 233 156 0 CDCP1_HUMANEEGVFTVTPDTK 239 157 0 CDCP1_HUMAN LSLVLVPAQK 241 157 157 CEAM8_HUMANLFIPNITTK 256 79 79 CEAM8_HUMAN TLTLLSVTR 257 79 0 CERU_HUMANGAYPLSIEPIGVR 258 158 0 CERU_HUMAN GPEEEHLGILGPVIWAEV 259 158 158 GDTIRCERU_HUMAN NNEGTYYSPNYNPQSR 261 158 0 CH1O_HUMAN GGEIQPVSVK 265 158 0CH1O_HUMAN VLLPEYGGTK 266 158 158 CLIC1_HUMAN FSAYIK 288 137 8CLIC1_HUMAN LAALNPESNTAGLDIFAK 290 137 129 CLIC1_HUMAN NSNPALNDNLEK 291137 0 CLUS_HUMAN ASSIIDELFQDR 293 158 0 CLUS_HUMAN EIQNAVNGVK 294 158158 CNTN1_HUMAN AHSDGGDGWSQVK 303 158 157 CNTN1_HUMAN DGEYWEVR 304 158 1CO6A3_HUMAN IGDLHPQIVNLLK 319 158 0 CO6A3_HUMAN VAVVQYSDR 321 158 158CO6A3_HUMAN WYYDPNTK 322 158 0 COF1_HUMAN EILVGDVGQTVDDPYA 328 127 0TFVK COF1_HUMAN LGGSAVISLEGKPL 329 127 0 COF1_HUMAN YALYDATYETK 330 127127 COIA1_HUMAN AVGLAGTFR 332 158 37 COIA1_HUMAN TEAPSATGQASSLLGGR 335158 121 CRP_HUMAN APLTKPLK 341 153 21 CRP_HUMAN ESDTSYVSLK 342 153 132CRP_HUMAN YEVQGEVFTKPQLWP 343 153 0 CSF1_HUMAN FNSVPLTDTGHER 351 134 113CSF1_HUMAN ISSLRPQGLSNPSTLSAQ 352 134 21 PQLSR CYTB_HUMAN SQLEEK 372 1000 CYTB_HUMAN SQVVAGTNYFIK 373 100 100 DESP_HUMAN YGDGIQLTR 384 131 131DMKN_HUMAN QVPGFGVADALGNR 395 128 0 DMKN_HUMAN VSEALGQGTR 397 128 128DSG2_HUMAN GQIIGNFQAFDEDTGLP 404 158 1 AHAR DSG2_HUMAN ILDVNDNIPVVENK405 158 157 EF1A1_HUMAN IGGIGTVPVGR 423 158 158 EF1A1_HUMAN QTVAVGVIK426 158 0 EF2_HUMAN FSVSPVVR 439 125 125 EF2_HUMAN GVQYLNEIK 441 125 0ENOA_HUMAN AVEHINK 452 156 0 ENOA_HUMAN YISPDQLADLYK 455 156 156ENOA_HUMAN YNQLLR 456 156 0 ENPL_HUMAN SGTSEFLNK 469 158 1 ENPL_HUMANSGYLLPDTK 470 158 157 EPHB6_HUMAN RPHFDQLVAAFDK 472 157 0 EPHB6_HUMANWAAPEVIAHGK 476 157 157 ERBB3_HUMAN GESIEPLDPSEK 483 105 0 ERBB3_HUMANLAEVPDLLEK 484 105 105 EREG_HUMAN VAQVSITK 487 115 115 EREG_HUMANVTSGDPELPQV 488 115 0 ERO1A_HUMAN AVLQWTK 489 121 0 ERO1A_HUMAN LLESDYFR491 121 0 ERO1A_HUMAN NLLQNIH 492 121 0 ERO1A_HUMAN VLPFFERPDFQLFTGNK493 121 121 F10A1_HUMAN AIDLFTDAIK 501 35 0 F10A1_HUMAN LQKPNAAIR 503 3535 FAM3C_HUMAN GINVALANGK 505 97 88 FAM3C_HUMAN SALDTAAR 507 97 9FAM3C_HUMAN TGEVLDTK 509 97 0 FCGR1_HUMAN HLEEELK 517 39 0 FCGR1_HUMANVFTEGEPLALR 519 39 39 FIBA_HUMAN GGSTSYGTGSETESPR 554 147 108 FIBA_HUMANNSLFEYQK 556 147 39 FINC_HUMAN SYTITGLQPGTDYK 561 154 135 FINC_HUMANVPGTSTSATLTGLTR 562 154 19 FKB11_HUMAN ANYWLK 565 23 0 FKB11_HUMANDPLVIELGQK 566 23 23 FOLH1_HUMAN GVILYSDPADYFAPGVK 569 138 0 FOLH1_HUMANLGSGNDFEVFFQR 570 138 138 FRIL_HUMAN DDVALEGVSHFFR 594 151 0 FRIL_HUMANLGGPEAGLGEYLFER 596 151 151 G3P_HUMAN GALQNIIPASTGAAK 599 150 149G3P_HUMAN LISWYDNEFGYSNR 600 150 1 G6PD_HUMAN DGLLPENTFIVGYAR 603 43 43G6PD_HUMAN GGYFDEFGIIR 604 43 0 G6PI_HUMAN AVLHVALR 608 39 6 G6PI_HUMANTLAQLNPESSLFIIASK 610 39 33 GDIR2_HUMAN DIVSGLK 629 158 158 GDIR2_HUMANLNYKPPPQK 630 158 0 GELS_HUMAN AQPVQVAEGSEPDGFWEA 634 158 0 LGGKGELS_HUMAN TASDFITK 637 158 158 GGH_HUMAN NLDGISHAPNAVK 640 158 158GGH_HUMAN YYIAASYVK 643 158 0 GRP78_HUMAN TWNDPSVQQDIK 664 158 90GRP78_HUMAN VYEGERPLTK 665 158 68 GSLG1_HUMAN IIIQESALDYR 666 158 158GSLG1_HUMAN LDPALQDK 667 158 0 GSLG1_HUMAN LIAQDYK 668 158 0 GSLG1_HUMANNDINILK 669 158 0 GSTP1_HUMAN ALPGQLKPFETLLSQNQ 672 123 123 GGKGSTP1_HUMAN YISLIYTNYEAGK 675 123 0 HPSE_HUMAN LPYPFSNK 714 49 0HPSE_HUMAN SVQLNGLTLK 715 49 49 HPT_HUMAN VGYVSGWGR 719 158 0 HPT_HUMANVTSIQDWVQK 720 158 158 HS90A_HUMAN SLTNDWEDHLAVK 724 32 32 HS90B_HUMANADHGEPIGR 726 121 0 HS90B_HUMAN IDIIPNPQER 728 121 121 HS90B_HUMANNPDDITQEEYGEFYK 730 121 0 HSPB1_HUMAN DGVVEITGK 732 30 0 HSPB1_HUMANGPSWDPFR 733 30 30 HTRA1_HUMAN LHRPPVIVLQR 741 40 40 HTRA1_HUMANLPVLLLGR 742 40 0 HTRA1_HUMAN VTAGISFAIPSDK 744 40 0 HXK1_HUMANFLLSESGSGK 747 117 17 HXK1_HUMAN LVDEYSLNAGK 749 117 47 HXK1_HUMANSANLVAATLGAILNR 750 117 53 HYOU1_HUMAN FPEHELTFDPQR 757 156 0HYOU1_HUMAN LPATEKPVLLSK 760 156 156 IBP2_HUMAN AEVLFR 762 158 0IBP2_HUMAN ELAVFR 763 158 158 IBP2_HUMAN LIQGAPTIR 765 158 0 IBP3_HUMANFHPLHSK 768 158 0 IBP3_HUMAN FLNVLSPR 769 158 0 IBP3_HUMAN YGQPLPGYTTK771 158 158 ICAM1_HUMAN ASVSVTAEDEGTQR 772 114 0 ICAM1_HUMANVELAPLPSWQPVGK 776 114 114 ICAM3_HUMAN IALETSLSK 780 158 0 ICAM3_HUMANTFVLPVTPPR 783 158 158 IF4A1_HUMAN GYDVIAQAQSGTGK 792 58 0 IF4A1_HUMANVLITTDLLAR 796 58 58 IGF1_HUMAN EGTEASLQIR 797 40 0 IGF1_HUMANISSLPTQLFK 798 40 40 IL18_HUMAN SDIIFFQR 807 45 45 IL18_HUMAN SVPGHDNK808 45 0 ILEU_HUMAN EATTNAPFR 824 88 13 ILEU_HUMAN TYNFLPEFLVSTQK 828 8875 ILK_HUMAN HSGIDFK 830 90 15 ILK_HUMAN QLNFLTK 832 90 75 ILK_HUMANWQGNDIVVK 833 90 0 INHBA_HUMAN AEVWLFLK 834 32 0 INHBA_HUMAN EGSDLSVVER835 32 32 ISLR_HUMAN ALPGTPVASSQPR 839 158 0 ISLR_HUMAN EVPLLQSLWLAHNEIR840 158 0 ISLR_HUMAN LPGLPEGAFR 841 158 158 ITA5_HUMAN SLQWFGATVR 846114 114 ITA5_HUMAN SSASSGPQILK 847 114 0 K1C18_HUMAN LASYLDR 861 72 0K1C18_HUMAN LQLETEIEALK 862 72 72 K1C18_HUMAN VVSETNDTK 863 72 0K1C19_HUMAN FGAQLAHIQALISGIEAQ 865 158 158 LGDVR K1C19_HUMAN FGPGVAFR866 158 0 KIT_HUMAN QATLTISSAR 876 158 158 KIT_HUMAN YVSELHLTR 878 158 0KLK14_HUMAN VLGSGTWPSAPK 889 27 27 KLK14_HUMAN VSGWGTISSPIAR 890 27 0KPYM_HUMAN APIIAVTR 899 158 13 KPYM_HUMAN LDIDSPPITAR 903 158 145LAMB2_HUMAN IQGTLQPHAR 910 69 0 LAMB2_HUMAN SLADVDAILAR 911 69 31LAMB2_HUMAN VLELSIPASAEQIQHLAG 913 69 38 AIAER LDHA_HUMAN FIIPNVVK 915157 0 LDHA_HUMAN LVIITAGAR 917 157 157 LDHB_HUMAN FIIPQIVK 920 158 157LDHB_HUMAN GLTSVINQK 921 158 1 LEG1_HUMAN GEVAPDAK 925 146 0 LEG1_HUMANLPDGYEFK 926 146 0 LEG1_HUMAN SFVLNLGK 927 146 146 LG3BP_HUMANASHEEVEGLVEK 938 158 158 LG3BP_HUMAN VEIFYR 941 158 0 LG3BP_HUMANYSSDYFQAPSDYR 942 158 0 LRP1_HUMAN TVLWPNGLSLDIPAGR 959 158 158LRP1_HUMAN VFFTDYGQIPK 960 158 0 LUM_HUMAN NIPTVNENLENYYLEVN 962 158 158QLEK LUM_HUMAN SLEDLQLTHNK 964 158 0 LYOX_HUMAN HWFQAGYSTSR 975 121 0LYOX_HUMAN TPILLIR 977 121 121 MASP1_HUMAN APGELEHGLITFSTR 991 158 151MASP1_HUMAN TGVITSPDFPNPYPK 994 158 7 MDHC_HUMAN LGVTANDVK 997 130 130MDHC_HUMAN VLVTGAAGQIAYSLLYSI 999 130 0 GNGSVFGK MDHM_HUMANVDFPQDQLTALTGR 1002 158 158 MDHM_HUMAN VSSFEEK 1004 158 0 MMP12_HUMANFLLILLLQATASGALP 1021 158 158 LNSSTSLEK MMP12_HUMAN GIQSLYGDPK 1022 1580 MMP12_HUMAN IDAVFYSK 1023 158 0 MMP2_HUMAN AFQVWSDVTPLR 1031 153 152MMP2_HUMAN IIGYTPDLDPETVDDA 1033 153 1 FAR MMP7_HUMAN LSQDDIK 1042 102102 MMP7_HUMAN NANSLEAK 1043 102 0 MMP9_HUMAN AFALWSAVTPLTFTR 1044 15850 MMP9_HUMAN FQTFEGDLK 1046 158 108 MMP9_HUMAN SLGPALLLLQK 1048 158 0MPRI_HUMAN GHQAFDVGQPR 1055 158 23 MPRI_HUMAN TYHSVGDSVLR 1056 158 4MPRI_HUMAN VPIDGPPIDIGR 1057 158 131 NCF4_HUMAN AEALFDFTGNSK 1095 138 43NCF4_HUMAN DAEGDLVR 1096 138 0 NCF4_HUMAN DIAVEEDLSSTPLLK 1097 138 0NCF4_HUMAN GATGIFPLSFVK 1098 138 95 NDKB_HUMAN DRPFFPGLVK 1105 24 0NDKB_HUMAN NIIHGSDSVK 1107 24 24 NRP1_HUMAN FVSDYETHGAGFSIR 1149 158 0NRP1_HUMAN FVTAVGTQGAISK 1150 158 158 NRP1_HUMAN SFEGNNNYDTPELR 1152 1580 OSTP_HUMAN AIPVAQDLNAPSDWDSR 1156 108 108 OSTP_HUMAN DSYETSQLDDQSAETH1157 108 0 SHK OSTP_HUMAN YPDAVATWLNPDPSQK 1160 108 0 PCBP2_HUMANIANPVEGSTDR 1189 52 0 PCBP2_HUMAN IITLAGPTNAIFK 1190 52 52 PCYOX_HUMANIAIIGAGIGGTSAAYYLR 1207 37 0 PCYOX_HUMAN IFSQETLTK 1208 37 37PCYOX_HUMAN TLLETLQK 1209 37 0 PDGFB_HUMAN SFDDLQR 1216 111 99PDGFB_HUMAN SHSGGELESLAR 1217 111 12 PDIA3_HUMAN ELSDFISYLQR 1225 129129 PDIA3_HUMAN SEPIPESNDGPVK 1227 129 0 PDIA4_HUMAN FDVSGYPTIK 1231 8181 PDIA4_HUMAN FHHTFSTEIAK 1232 81 0 PECA1_HUMAN SELVTVTESFSTPK 1241 770 PECA1_HUMAN STESYFIPEVR 1242 77 77 PEDF_HUMAN LQSLFDSPDFSK 1246 158 0PEDF_HUMAN TVQAVLTVPK 1248 158 158 PGAM1_HUMAN HGESAWNLENR 1259 14 14PLIN2_HUMAN DAVTTTVTGAK 1264 138 0 PLIN2_HUMAN EVSDSLLTSSK 1265 138 138PLSL_HUMAN IGNFSTDIK 1284 158 0 PLSL_HUMAN ISFDEFIK 1285 158 158PLXB3_HUMAN ELPVPIYVTQGEAQR 1294 77 0 PLXB3_HUMAN GPVDAVTGK 1296 77 77PLXC1_HUMAN FWVNILK 1299 158 0 PLXC1_HUMAN LNTIGHYEISNGSTIK 1300 158 158POSTN_HUMAN GFEPGVTNILK 1302 158 158 POSTN_HUMAN IIDGVPVEITEK 1303 158 0POSTN_HUMAN IIHGNQIATNGVVHVIDR 1304 158 0 PPIB_HUMAN VIFGLFGK 1319 158 0PPIB_HUMAN VYFDLR 1320 158 158 PRDX1_HUMAN IGHPAPNFK 1325 158 116PRDX1_HUMAN QITVNDLPVGR 1328 158 42 PROF1_HUMAN STGGAPTFNVTVTK 1338 158157 PROF1_HUMAN TFVNITPAEVGVLVGK 1339 158 1 PRS6A_HUMAN VDILDPALLR 134913 13 PTGIS_HUMAN DPEIYTDPEVFK 1352 158 0 PTGIS_HUMAN LLLFPFLSPQR 1357158 158 PTPA_HUMAN FGSLLPIHPVTSG 1361 103 103 PTPA_HUMANTGPFAEHSNQLWNISAVP 1363 103 0 SWSK PTPA_HUMAN VDDQIAIVFK 1364 103 0PTPA_HUMAN WIDETPPVDQPSR 1365 103 0 PTPRJ_HUMAN AVSISPTNVILTWK 1372 1580 PTPRJ_HUMAN VITEPIPVSDLR 1374 158 158 PVR_HUMAN SVDIWLR 1379 158 158PVR_HUMAN VLAKPQNTAEVQK 1380 158 0 RAB32_HUMAN VHLPNGSPIPAVLLANK 1384 220 RAB32_HUMAN VLVIGELGVGK 1385 22 22 RAN_HUMAN FNVWDTAGQEK 1391 116 2RAN_HUMAN LVLVGDGGTGK 1392 116 114 RAN_HUMAN NVPNWHR 1393 116 0RAP2B_HUMAN EVSYGEGK 1395 145 0 RAP2B_HUMAN VDLEGER 1397 145 145S10A1_HUMAN DVDAVDK 1408 128 128 S10A1_HUMAN ELLQTELSGFLDAQK 1409 128 0S10A6_HUMAN ELTIGSK 1411 154 154 S10A6_HUMAN LQDAEIAR 1412 154 0SAA_HUMAN EANYIGSDK 1414 143 0 SAA_HUMAN SFFSFLGEAFDGAR 1416 143 143SCF_HUMAN LFTPEEFFR 1418 143 143 SCF_HUMAN LVANLPK 1419 143 0SEM3G_HUMAN DYPDEVLQFAR 1426 155 0 SEM3G_HUMAN LFLGGLDALYSLR 1428 155155 SIAL_HUMAN AYEDEYSYFK 1449 19 19 SIAL_HUMAN TTSPPFGK 1452 19 0SODM_HUMAN GDVTAQIALQPALK 1460 154 151 SODM_HUMAN NVRPDYLK 1462 154 3SPON2_HUMAN WSQTAFPK 1478 63 0 SPON2_HUMAN YSITFTGK 1479 63 63STAT1_HUMAN TELISVSEVHPSR 1494 38 29 STAT1_HUMAN YTYEHDPITK 1496 38 9TBA1B_HUMAN AVFVDLEPTVIDEVR 1519 119 119 TBA1B_HUMAN EIIDLVLDR 1520 1190 TBB3_HUMAN ISVYYNEASSHK 1533 158 158 TBB3_HUMAN YLTVATVFR 1535 158 0TCPA_HUMAN IHPTSVISGYR 1540 158 4 TCPA_HUMAN SSLGPVGLDK 1542 158 154TCPQ_HUMAN DIDEVSSLLR 1550 48 0 TCPQ_HUMAN NVGLDIEAEVPAVK 1553 48 48TCPZ_HUMAN GIDPFSLDALSK 1557 6 6 TCPZ_HUMAN GLVLDHGAR 1558 6 0TENA_HUMAN GLEPGQEYNVLLTAEK 1570 140 140 TENA_HUMAN TVSGNTVEYALTDLEPA1572 140 0 TEYTLR TENX_HUMAN DAQGQPQAVPVSGDLR 1574 158 158 TENX_HUMANYEVTVVSVR 1578 158 0 TERA_HUMAN GILLYGPPGTGK 1579 106 94 TERA_HUMANLDQLIYIPLPDEK 1582 106 12 TETN_HUMAN GGTLSTPQTGSENDALY 1588 158 118 EYLRTETN_HUMAN LDTLAQEVALLK 1589 158 40 TFR1_HUMAN LTVSNVLK 1598 157 0TFR1_HUMAN SSGLPNIPVQTISR 1600 157 157 TIMP1_HUMAN GFQALGDAADIR 1610 151151 TIMP1_HUMAN SEEFLIAGK 1611 151 0 TNF12_HUMAN AAPFLTYFGLFQVH 1621 156156 TNF12_HUMAN INSSSPLR 1622 156 0 TPIS_HUMAN VVFEQTK 1634 157 157TPIS_HUMAN VVLAYEPVWAIGTGK 1635 157 0 TRFL_HUMAN FQLFGSPSGQK 1637 48 22TRFL_HUMAN LRPVAAEVYGTER 1638 48 4 TRFL_HUMAN VPSHAVVAR 1639 48 5TRFL_HUMAN YYGYTGAFR 1640 48 17 TSP1_HUMAN GFLLLASLR 1644 158 6TSP1_HUMAN GTSQNDPNWVVR 1645 158 152 TTHY_HUMAN TSESGELHGLTTEEEFV 164627 27 EGIYK TTHY_HUMAN VEIDTK 1647 27 0 TYPH_HUMAN ALQEALVLSDR 1648 59 0TYPH_HUMAN TLVGVGASLGLR 1651 59 59 UGGG1_HUMAN DLSQNFPTK 1653 58 58UGGG1_HUMAN FTILDSQGK 1654 58 0 UGPA_HUMAN LVEIAQVPK 1669 97 97UGPA_HUMAN NENTFLDLTVQQIEHLNK 1670 97 0 VA0D1_HUMAN LLFEGAGSNPGDK 167913 13 VA0D1_HUMAN NVADYYPEYK 1681 13 0 VEGFC_HUMAN DLEEQLR 1689 21 21VEGFC_HUMAN EAPAAAAAFESGLDLSDA 1690 21 0 EPDAGEATAYASK VEGFC_HUMANFAAAHYNTEILK 1692 21 0 VEGFC_HUMAN NQPLNPGK 1693 21 0 VTNC_HUMANAVRPGYPK 1705 145 0 VTNC_HUMAN DVWGIEGPIDAAFTR 1707 145 145 ZA2G_HUMANEIPAWVPFDPAAQITK 1724 158 158 ZA2G_HUMAN WEAEPVYVQR 1725 158 0

Exemplary Biomarker Protein

The following 36 proteins were identified as biomarker candidates in alarge-scale experiment of 72 lung cancer samples and 71 benign lungnodule samples using the MS-LC-SRM-MS system described herein.

TABLE 4 Coeff- icient Coef- Pre- (Discov- ficient dicted Official Coop-Coef- ery) (Final) Concen- Protein Gene erative Partial ficient SEQ IDalpha = alpha = Tissue tration Category (UniProt) Name Score AUC CVTransition NO: 36.16 26.25 Candidate (ng/ml) Classifier TSP1_ THBS1 1.80.25 0.24 GFLLLASLR_ 1644 0.53 0.44 510 HUMAN 495.31_559.40 ClassifierCOIA1_ COL18A1 3.7 0.16 0.25 AVGLAGTFR_ 332 −1.56 −0.91 35 HUMAN446.26_721.40 Classifier ISLR_ ISLR 1.4 0.32 0.25 ALPGTPVASSQPR_ 8391.40 0.83 — HUMAN 640.85_841.50 Classifier TETN_ CLEC3B 2.5 0.26 0.26LDTLAQEVALLK_ 1589 −1.79 −1.02 58000 HUMAN 657.39_330.20 ClassifierFRIL_ FTL 2.8 0.31 0.26 LGGPEAGLGEYLFER_ 596 0.39 0.17 Secreted, 12HUMAN 804.40_913.40 Epi, Endo Classifier GRP78_ HSPA5 1.4 0.27 0.27TWNDPSVQQDIK_ 664 1.41 0.55 Secreted, 100 HUMAN 715.85_260.20 Epi, EndoClassifier ALDOA_ ALDOA 1.3 0.26 0.28 ALQASALK_ 58 −0.80 −0.26 Secreted,250 HUMAN 401.25_617.40 Epi Classifier BGH3_ TGFBI 1.8 0.21 0.28LTLLAPLNSVFK_ 139 1.73 0.54 Epi 140 HUMAN 658.40_804.50 ClassifierLG3BP_ LGALS3BP 4.3 0.29 0.29 VEIFYR_413.73_ 941 −0.58 −0.21 Secreted440 HUMAN 598.30 Classifier LRP1_ LRP1 4.0 0.13 0.32 TVLWPNGLSLDIPAGR_959 −1.59 −0.83 Epi 20 HUMAN 855.00_400.20 Classifier FIBA_ FGA 1.1 0.310.35 NSLFEYQK_514.76 556 0.31 0.13 130000 HUMAN 714.30 Classifier PRDX1_PRDX1 1.5 0.32 0.37 QITVNDLPVGR_ 1328 −0.34 −0.26 Epi 60 HUMAN606.30_428.30 Classifier GSLG1_ GLG1 1.2 0.34 0.45 IIIQESALDYR_ 666−0.70 −0.44 Epi, — HUMAN 660.86_338.20 Endo Robust KIT_ KIT 1.4 0.330.46 8.2 HUMAN Robust CD14_ CD14 4.0 0.33 0.48 Epi 420 HUMAN RobustEF1A1_ EEF1A1 1.2 0.32 0.56 Secreted, 61 HUMAN Epi Robust TENX_ TNXB 1.10.30 0.56 Endo 70 HUMAN Robust AIFM1_ AIFM1 1.4 0.32 0.70 Epi, 1.4 HUMANEndo Robust GGH_ GGH 1.3 0.32 0.81 250 HUMAN Robust IBP3_ IGFBP3 3.40.32 1.82 5700 HUMAN Robust ENPL_ HSP90B1 1.1 0.29 5.90 Secreted, 88HUMAN Epi, Endo Non- ERO1A_ ERO1L 6.2 Secreted, — Robust HUMAN Epi, EndoNon- 6PGD_ PGD 4.3 Epi, 29 Robust HUMAN Endo Non- ICAM1_ ICAM1 3.9 71Robust HUMAN Non- PTPA_ PPP2R4 2.1 Endo 3.3 Robust HUMAN Non- NCF4_ NCF42.0 Endo — Robust HUMAN Non- SEM3G_ SEMA3G 1.9 — Robust HUMAN Non-1433T_ YWHAQ 1.5 Epi 180 Robust HUMAN Non- RAP2B_ RAP2B 1.5 Epi — RobustHUMAN Non- MMP9_ MMP9 1.4 28 Robust HUMAN Non- FOLH1_ FOLH1 1.3 — RobustHUMAN Non- GSTP1_ GSTP1 1.3 Endo 32 Robust HUMAN Non- EF2_ EEF2 1.3Secreted, 30 Robust HUMAN Epi Non- RAN_ RAN 1.2 Secreted, 4.6 RobustHUMAN Epi Non- SODM_ SOD2 1.2 Secreted 7.1 Robust HUMAN Non- DSG2_ DSG21.1 Endo 2.7 Robust HUMAN

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20150323545A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. A composition comprising isolated synthetic peptides said compositioncomprising ALQASALK (SEQ ID NO: 58), GFLLLASLR (SEQ ID NO: 1644),LGGPEAGLGEYLFER (SEQ ID NO: 596), YVSELHLTR (SEQ ID NO: 878), orYYIAASYVK (SEQ ID NO: 643).
 2. A composition comprising at least 10isolated synthetic peptides said composition comprising at least 2isolated synthetic peptides of at least 5 proteins selected from LRP1,BGH3, COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA,KIT, GGH or GSLG1, and wherein the at least two isolated syntheticpeptides are selected from ADDGRPFPQVIK (SEQ ID NO: 57), ALQASALK (SEQID NO: 58), QLLLTADDR (SEQ ID NO: 60), ADHHATNGVVHLIDK (SEQ ID NO: 136),DILATNGVIHYIDELLIPDSAK (SEQ ID NO: 137), LTLLAPLNSVFK (SEQ ID NO: 139),SPYQLVLQHSR (SEQ ID NO: 140), ADDILASPPR (SEQ ID NO: 331), AVGLAGTFR(SEQ ID NO: 332), TEAPSATGQASSLLGGR (SEQ ID NO: 335), GGSTSYGTGSETESPR(SEQ ID NO 554), NSLFEYQK (SEQ ID NO: 556), TVIGPDGHK (SEQ ID NO: 557),VQHIQLLQK (SEQ ID NO: 558), ALFQDIK (SEQ ID NO: 593), DDVALEGVSHFFR (SEQID NO: 594), KPAEDEWGK (SEQ ID NO: 595), LGGPEAGLGEYLFER (SEQ ID NO:596), LNQALLDLHALGSAR (SEQ ID NO: 597), MGDHLTNLHR (SEQ ID NO: 598),LYGSAGPPPTGEEDTAEK (SEQ ID NO: 663), TWNDPSVQQDIK (SEQ ID NO: 664),VYEGERPLTK (SEQ ID NO: 665), IIIQESALDYR (SEQ ID NO: 666), LDPALQDK (SEQID NO: 667), LIAQDYK (SEQ ID NO: 668), NDINILK (SEQ ID NO: 669),VAELSSDDFHLDR (SEQ ID NO: 670), ALPGTPVASSQPR (SEQ ID NO: 839),EVPLLQSLWLAHNEIR (SEQ ID NO: 840), LPGLPEGAFR (SEQ ID NO: 841),TVAAGALASLSHLK (SEQ ID NO: 843), ASHEEVEGLVEK (SEQ ID NO: 938),STHTLDLSR (SEQ ID NO: 940), VEIFYR (SEQ ID NO: 941), YSSDYFQAPSDYR (SEQID NO: 942), AALSGANVLTLIEK (SEQ ID NO: 958), TVLWPNGLSLDIPAGR (SEQ IDNO: 959), VFFTDYGQIPK (SEQ ID NO: 960), ADEGISFR (SEQ ID NO: 1321),ATAVMPDGQFK (SEQ ID NO: 1322), DISLSDYK (SEQ ID NO: 1323), GLFIIDDK (SEQID NO: 1324), IGHPAPNFK (SEQ ID NO: 1325), LVQAFQFTDK (SEQ ID NO: 1326),QGGLGPMNIPLVSDPK (SEQ ID NO: 1327), QITVNDLPVGR (SEQ ID NO: 1328),SVDETLR (SEQ ID NO: 1329), TIAQDYGVLK (SEQ ID NO: 1330),GGTLSTPQTGSENDALYEYLR (SEQ ID NO: 1588), LDTLAQEVALLK (SEQ ID NO: 1589),NWETEITAQPDGGK (SEQ ID NO: 1590), FQDLVDAVR (SEQ ID NO: 1641),FTGSQPFGQGVEHATANK (SEQ ID NO: 1642), FVFGTIPEDILR (SEQ ID NO: 1643),GFLLLASLR (SEQ ID NO: 1644), YVSELHLTR (SEQ ID NO: 878), YYIAASYVK (SEQID NO: 643) or GTSQNDPNWVVR (SEQ ID NO: 1645).
 3. The composition ofclaim 2, wherein at least one of the isolated synthetic peptides isisotopically labeled.
 4. The composition of claim 2, wherein themeasured quantity of each of the at least 2 isolated synthetic peptidesof at least 5 proteins is known.
 5. The composition of claim 2, furthercomprising one or more polar solvents.
 6. The composition of claim 2,further comprising an additional five isolated synthetic peptides of atleast one protein selected from LRP1, BGH3, COIA1, TETN, TSP1, ALDOA,GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, KIT, GGH or GSLG1.
 7. Thecomposition of claim 2, further comprising an additional five isolatedsynthetic peptides of at least one protein selected from APOE, BASP1,CD14, FOXA2 or HSPB1.
 8. A composition comprising at least 25 isolatedsynthetic peptides said composition comprising at least five isolatedsynthetic peptides of at least five proteins selected from LRP1, BGH3,COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, GSLG1,KIT or GGH wherein the at least five isolated synthetic peptides areselected from ADDGRPFPQVIK (SEQ ID NO: 57), ALQASALK (SEQ ID NO: 58),QLLLTADDR (SEQ ID NO: 60), ADHHATNGVVHLIDK (SEQ ID NO: 136),DILATNGVIHYIDELLIPDSAK (SEQ ID NO: 137), LTLLAPLNSVFK (SEQ ID NO: 139),SPYQLVLQHSR (SEQ ID NO: 140), ADDILASPPR (SEQ ID NO: 331), AVGLAGTFR(SEQ ID NO: 332), TEAPSATGQASSLLGGR (SEQ ID NO: 335), GGSTSYGTGSETESPR(SEQ ID NO 554), NSLFEYQK (SEQ ID NO: 556), TVIGPDGHK (SEQ ID NO: 557),VQHIQLLQK (SEQ ID NO: 558), ALFQDIK (SEQ ID NO: 593), DDVALEGVSHFFR (SEQID NO: 594), KPAEDEWGK (SEQ ID NO: 595), LGGPEAGLGEYLFER (SEQ ID NO:596), LNQALLDLHALGSAR (SEQ ID NO: 597), MGDHLTNLHR (SEQ ID NO: 598),LYGSAGPPPTGEEDTAEK (SEQ ID NO: 663), TWNDPSVQQDIK (SEQ ID NO: 664),VYEGERPLTK (SEQ ID NO: 665), IIIQESALDYR (SEQ ID NO: 666), LDPALQDK (SEQID NO: 667), LIAQDYK (SEQ ID NO: 668), NDINILK (SEQ ID NO: 669),VAELSSDDFHLDR (SEQ ID NO: 670), ALPGTPVASSQPR (SEQ ID NO: 839),EVPLLQSLWLAHNEIR (SEQ ID NO: 840), LPGLPEGAFR (SEQ ID NO: 841),TVAAGALASLSHLK (SEQ ID NO: 843), ASHEEVEGLVEK (SEQ ID NO: 938),STHTLDLSR (SEQ ID NO: 940), VEIFYR (SEQ ID NO: 941), YSSDYFQAPSDYR (SEQID NO: 942), AALSGANVLTLIEK (SEQ ID NO: 958), TVLWPNGLSLDIPAGR (SEQ IDNO: 959), VFFTDYGQIPK (SEQ ID NO: 960), ADEGISFR (SEQ ID NO: 1321),ATAVMPDGQFK (SEQ ID NO: 1322), DISLSDYK (SEQ ID NO: 1323), GLFIIDDK (SEQID NO: 1324), IGHPAPNFK (SEQ ID NO: 1325), LVQAFQFTDK (SEQ ID NO: 1326),QGGLGPMNIPLVSDPK (SEQ ID NO: 1327), QITVNDLPVGR (SEQ ID NO: 1328),SVDETLR (SEQ ID NO: 1329), TIAQDYGVLK (SEQ ID NO: 1330),GGTLSTPQTGSENDALYEYLR (SEQ ID NO: 1588), LDTLAQEVALLK (SEQ ID NO: 1589),NWETEITAQPDGGK (SEQ ID NO: 1590), FQDLVDAVR (SEQ ID NO: 1641),FTGSQPFGQGVEHATANK (SEQ ID NO: 1642), FVFGTIPEDILR (SEQ ID NO: 1643),GFLLLASLR (SEQ ID NO: 1644), YYIAASYVK (SEQ ID NO: 643), YVSELHLTR (SEQID NO: 878), or GTSQNDPNWVVR (SEQ ID NO: 1645).
 9. The composition ofclaim 8, wherein the at least five isolated synthetic peptides areselected from GFLLLASLR (SEQ ID NO: 1644), AVGLAGTFR (SEQ ID NO: 332),ALPGTPVASSQPR (SEQ ID NO: 839), LDTLAQEVALLK (SEQ ID NO: 1589),LGGPEAGLGEYLFER (SEQ ID NO: 596), TWNDPSVQQDIK (SEQ ID NO: 664),ALQASALK (SEQ ID NO: 58), LTLLAPLNSVFK (SEQ ID NO: 139), VEIFYR (SEQ IDNO: 941), TVLWPNGLSLDIPAGR (SEQ ID NO: 959), NSLFEYQK (SEQ ID NO: 556),QITVNDLPVGR (SEQ ID NO: 1328), YYIAASYVK (SEQ ID NO: 643), YVSELHLTR(SEQ ID NO: 878), and IIIQESALDYR (SEQ ID NO: 666).
 10. A compositioncomprising at least 5 transition ion pairs said composition comprisingat least one transition ion pair of at least 5 proteins selected fromALDOA, TSP1, FRIL, KIT, and GGH, wherein the at least one transition ionpair consists of a precursor ion with a corresponding m/z and a fragmention with a corresponding ion m/z, and wherein the transition ion pairsare selected from precursor ALQASALK (SEQ ID NO: 58) transition pair401.25-260.2, 401.25-331.2, 401.25-489.3, or 401.25-617.4; precursorGFLLLASLR (SEQ ID NO: 1644) transition pair 495.31-318.2, 495.31-375.2,495.31-446.3, or 495.31-559.4; precursor YYIAASYVK (SEQ ID NO: 643)transition pair 539.28-567.3, 539.28-638.40, 539.28-751.4, or539.28-914.5; precursor LGGPEAGLGEYLFER (SEQ ID NO: 596) transition pair804.41-451.2, 804.41-525.3, 804.41-564.3, or 804.41-913.4; and precursorYVSELHLTR (SEQ ID NO: 878) transition pair 373.21-263.1, 373.21-276.2,373.21-526.3, or 373.21-639.4, wherein each of the precursor ions has adouble or triple charge.
 11. The composition of claim 10, furthercomprising an additional five transition ion pairs comprising onetransition ion pair of at least one protein selected from LRP1, BGH3,COIA1, TETN, TSP1, ALDOA, GRP78, ISLR, FRIL, LG3BP, PRDX1, FIBA, KIT,GGH or GSLG1.
 12. The composition of claim 11, further comprising anadditional five transition ion pairs comprising one transition ion pairof at least one protein selected from APOE, BASP1, CD14, FOXA2 or HSPB1,wherein the additional one transition ion pair consists of a precursorion with a corresponding m/z and a fragment ion with a correspondingm/z, and wherein the transition ion pairs are selected from precursorAATVGSLAGQPLQER (SEQ ID NO: 82) transition pair 479.4-600.3,479.4-642.4, 479.4-827.4, or 479.4-898.5; precursor ALMDETMK (SEQ ID NO:83) transition pair 469.72-508.2, 469.72-469.72, 469.72-508.2,469.72-623.3, 469.72-754.3, or 469.72-867.4; precursor GEVQAMLGQSTEELR(SEQ ID NO: 85) transition pair 824.4-616.3, 824.4-729.4, 824.4-734.4,or 824.4-919.4; precursor LAVYQAGAR (SEQ ID NO: 86) transition pair474.77-502.3, 474.77-665.3, 474.77-764.4, or 474.77-835.4; precursorLGPLVEQGR (SEQ ID NO: 87) transition pair 484.78-588.3, 484.78-701.4,484.78-798.4, or 484.78-855.5; precursor SELEEQLTPVAEETR (SEQ ID NO: 88)transition pair 865.93-459.2, 865.93-801.4, 865.93-902.5, or865.93-930.4; precursor SWFEPLVEDMQR (SEQ ID NO: 89) transition pair768.86-274.1, 768.86-421.2, 768.86-678.3, or 768.86-987.5; precursorWELALGR (SEQ ID NO: 90) transition pair 422.74-316.1, 422.74-345.2,422.74-529.3, or 422.74-658.4; precursor AAEAAAAPAESAAPAAGEEPSK (SEQ IDNO: 118) transition pair 983.97-414.2, 983.97-485.2, 983.97-556.3, or983.97-885.4; precursor AEGAATEEEGTPK (SEQ ID NO: 119) transition pair645.3-258.1, 645.3-789.4, 645.3-890.4, or 645.3-961.4; precursorAPEQEQAAPGPAAGGEAPK (SEQ ID NO: 120) transition pair 592.62-629.3,592.62-754.3, 592.62-825.4, or 592.62-951.5; precursor EKPDQDAEGK (SEQID NO: 122) transition pair 558.76-404.2, 558.76-647.3, 558.76-859.4, or558.76-913.4; precursor ESEPQAAAEPAEAK (SEQ ID NO: 123) transition pair714.34-515.3, 714.34-644.3, 714.34-786.4, or 714.34-857.4; precursorETPAATEAPSSTPK (SEQ ID NO: 124) transition pair 693.84-616.3,693.84-816.4, 693.84-917.5, or 693.84-988.5; precursor GYNVNDEK (SEQ IDNO: 125) transition pair 469.71-276.2, 469.71-505.2, 469.71-604.3, or469.71-718.3; precursor SDGAPASDSKPGSSEAAPSSK (SEQ ID NO: 126)transition pair 644.96-260.1, 644.96-331.1, 644.96-418.2, or644.96-586.2; precursor TEAPAAPAAQETK (SEQ ID NO: 127) transition pair642.83-541.3, 642.83-744.4, 642.83-815.4, or 642.83-983.5; precursorAFPALTSLDLSDNPGLGER (SEQ ID NO: 206) transition pair 987-500.3,987-628.3, 987-742.4, or 987-944.4; precursor ATVNPSAPR (SEQ ID NO: 207)transition pair 456.75-386.2, 456.75-430.2, 456.75-527.3, or456.75-740.4; precursor ELTLEDLK (SEQ ID NO: 208) transition pair480.77-260.2, 480.77-504.3, 480.77-617.4, or 480.77-718.4; precursorFPAIQNLALR (SEQ ID NO: 209) transition pair 571.84-714.4, 571.84-827.5,571.84-898.5, or 571.84-995.6; precursor LTVGAAQVPAQLLVGALR (SEQ ID NO:211) transition pair 593.03-416.3, 593.03-515.3, 593.03-628.4, or593.03-740.4; precursor NVSWATGR (SEQ ID NO: 212) transition pair445.73-301.2, 445.73-404.2, 5, 445.73-590.3, or 445.73-677.3; precursorSTLSVGVSGTLVLLQGAR (SEQ ID NO: 213) transition pair 879.51-431.2,879.51-544.3, 879.51-657.4, or 879.51-970.6; precursor SWLAELQQWLKPGLK(SEQ ID NO: 214) transition pair 599.67-274.1, 599.67-414.3,599.67-542.4, or 599.67-841.5; precursor VDADADPR (SEQ ID NO: 215)transition pair 429.7-272.2, 429.7-573.3, 429.7-644.3, or 429.7-759.3;precursor AYEQVMHYPGYGSPMPGSLAMGPVTNK (SEQ ID NO: 577) transition pair961.45-558.3, 961.45-615.3, 961.45-817.4, or 961.45-930.5; precursorEAAGAAGSGK (SEQ ID NO: 578) transition pair 409.7-348.2, 409.7-490.3,409.7-547.3, or 409.7-618.3; precursor MHSASSMLGAVK (SEQ ID NO: 579)transition pair 406.87-374.2, 406.87-487.3, 406.87-601.2, or406.87-732.3; precursor TGLDASPLAADTSYYQGVYSRPIMNSS (SEQ ID NO: 582)transition pair 955.45-387.2, 955.45-545.3, 955.45-648.3, or955.45-826.4; precursor AQLGGPEAAK (SEQ ID NO: 731) transition pair471.26-313.2, 471.26-427.2, 471.26-629.3, or 471.26-742.4; precursorDGVVEITGK (SEQ ID NO: 732) transition pair 459.25-305.2, 459.25-418.3,459.25-547.3, or 459.25-646.4; precursor GPSWDPFR (SEQ ID NO: 733)transition pair 481.23-419.2, 481.23-534.3, 481.23-720.3, or481.23-807.4; precursor LATQSNEITIPVTFESR (SEQ ID NO: 734) transitionpair 953.5-639.3, 953.5-744.4, 953.5-835.4, or 953.5-857.4; precursorLFDQAFGLPR (SEQ ID NO: 735) transition pair 582.31-272.2; 582.31-442.3,582.31-660.4, or 582.31-903.5; precursor QDEHGYISR (SEQ ID NO: 736)transition pair 368.84-262.2, 368.84-538.3, 368.84-595.3, 368.84-730.3,368.84-504.3, 368.84-660.4, 368.84-747.4, or 368.84-834.4; precursorVPFSLLR (SEQ ID NO: 738) transition pair 416.26-488.3, 416.26-488.3,416.26-635.4, or 416.26-732.4; or precursor VSLDVNHFAPDELTVK (SEQ ID NO:739) transition pair 595.31-347.2, 595.31-460.3, 595.31-801.4, or595.31-872.5, wherein each of the precursor ions has a double or triplecharge.
 13. The composition of claim 11, wherein the at least fivetransition ion pairs are selected from precursor GFLLLASLR (SEQ ID NO:1644) transition pair 495.31-559.40, precursor LGGPEAGLGEYLFER (SEQ IDNO: 596) transition pair 804.40-913.40, precursor ALQASALK (SEQ ID NO:58) transition pair 401.25-617.40, YYIAASYVK (SEQ ID NO: 643) transitionpair 539.28-638.40, precursor LGGPEAGLGEYLFER (SEQ ID NO: 596)transition pair 804.41-451.2, 804.41-525.3, 804.41-564.3, 804.41-913.4,and precursor YVSELHLTR (SEQ ID NO: 878) transition pair 373.21-263.1,373.21-276.2, 373.21-526.3, 373.21-639.4.
 14. A method comprisinganalyzing the composition of claim 8 using mass spectrometry.
 15. Themethod of claim 14, wherein the method uses selected reaction monitoringmass spectrometry.
 16. A method comprising analyzing the composition ofclaim 8 using Enzyme-Linked Immunosorbent Assay (ELISA).
 17. Amultiplexed LC-SRM-MS assay for the measurement of a plurality ofproteins in a single sample comprising: a) generating a set of peptidesand corresponding transitions for each protein to be monitored; b)optimizing the collision energy for each transition such thatinterference among the transitions is avoided; c) selecting a set oftransitions that have the greatest peak areas for each protein, andwherein the selected transitions do not interfere with the ions in thesample; d) measuring a plurality of proteins in a sample.
 18. The assayaccording to claim 17 wherein the two best peptides per protein and thetwo best transitions per peptide are selected based on experimental dataresulting from LC-SRM-MS analysis of one or more of the followingexperimental samples: a biological disease sample, a biological controlsample, and a mixture of synthetic peptides.
 19. The assay according toclaim 18 wherein the biological disease and biological control samplesare processed using an immunodepletion method prior to LC-SRM-MSanalysis.
 20. The assay according to claim 19, wherein the experimentalsamples contain internal standard peptides.
 21. The assay according toclaim 19, wherein the LC-SRM-MS analysis method specifies a maximum of7000 transitions, including transitions of the internal standardpeptides and transitions.
 22. The Assay according to claim 17, whereinthe top two transitions per peptide are selected according to one ormore of the following criteria: (1) the transitions exhibit the largestpeak areas measured in either of the two biological samples; (2) thetransitions are not interfered with by other ions; (3) the transitionsdo not exhibit an elution profile that visually differs from those ofother transitions of the same peptide; (4) the transitions are notbeyond the detection limit of both of the two biological experimentalsamples; and (5) the transitions do not exhibit interferences.
 23. Theassay according to claim 17, wherein the top two peptides per proteinare selected according to one or more of the following criteria: (1) oneor more peptides exhibit two transitions according to claim 20 andrepresent the largest combined peak areas of the two transitionsaccording to claim 20; and (2) one or more peptides exhibit onetransition according to claim 20 and represent the largest combined peakareas of the two transitions according to claim
 20. 24. An assaydeveloped according to claim 17.